Cancer stem cell antigen vaccines and methods

ABSTRACT

Method of stimulating an immune response (e.g., to treat cancer) include administering to a patient a composition including dendritic cells that present cancer stem cell antigens. Compositions including cancer stem cell antigens are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/826,955, filed on Sep. 26, 2006, theentire contents of which are incorporated herein by reference.

BACKGROUND

Over the past 30 years, a wealth of information has been generatedconcerning the in vivo and in vitro properties of brain tumors androdent models of brain tumor. The 9L gliosarcoma, which was generatedfrom inbred Fisher rats, is a widely used syngeneic rat model for braintumors. Originally produced by N-methyl-nitrosourea mutagenesis inFisher rats, the tumor was cloned and designated 9L gliosarcoma becauseof its dual appearance of a glioblastoma and a sarcoma (Benda et al., J.Neurosurg., 34:310-323, 1971; Schmidek et al., J. Neurosurg.,34:335-340, 1971). The tumor could be proliferated under in vivo and invitro conditions, making it a useful candidate as a glioma tumor model.The 9L gliosarcoma model clinically mimics rapidly growing and fatalintracerebral tumors, making it the most widely used rat brain tumormodel.

Stem cells have been defined as multipotent, self-renewing cells withthe potential to differentiate into multiple cell types. Systems havebeen developed to identify the first neural stein cells in a definedmedia, whereby striatal embryonic progenitors could be harvested andgrown in culture as undifferentiated neurospheres (clonally derivedaggregates of cells derived from a single stem cell) under the influenceof the mitogens EGF and bFGF. Many of these cells expressed nestin (anintermediate filament found in neuroepithelial stem cells), but notmarkers for the more differentiated principal cell types of theCNS—neuronal and glial cells. However, when grown on coverslips treatedwith poly-L-ornithine, a substrate that allows cellular adhesion, manyof the cells within the neurospheres differentiated into neurons andastrocytes with discontinued nestin expression. The isolated striatalcells fulfilled the critical features expected from neural stem cells:an unlimited capacity for self-renewal and capacity to differentiateinto the principal mature neural cells (Potten et al., Development,110:1001-1020, 1990; Lee et al., Nat. Neurosci., 8:723-729, 2005; Maricet al., J. Neurosci., 23:240-251, 2003; Weissman et al., Annu. Rev.Cell. Dev. Biol., 17:387-403, 2001; Seaberg et al., Trends Neurosci.,26:125-131, 2003; Reya et al., Nature, 414:105-111, 2001). Under similarcircumstances “cancer stem cells” appear to have the samecharacteristics of self-renewal and multipotentcy.

Malignant brain tumors carry a poor prognosis even in the midst ofsurgical, radio-, and chemotherapy. With the poor prognosis of braintumors amidst the available therapeutic treatments, there exists asignificant need for more effective therapies to treat such tumors.

SUMMARY

This invention is based, inter alia, on the discovery that vaccinesbased on cancer stem cell antigens are exceptionally useful for therapyof cancer. Immunization of animals with dendritic cells pulsed withantigens from isolated cancer stem cells provided a significant survivalbenefit as compared to immunization with dendritic cells pulsed withdifferentiated tumor cells. Cancer stem cells were found to expressmajor histocompatibility (MHC), indicating that they can displayantigens. Further, proteins differentially expressed in cancer stemcells as compared to differentiated tumor cells were identified. Theseproteins can be useful in providing antigenic compositions for treatmentof cancers (e.g., neural cancers such as gliomas).

Accordingly, this application provides methods and compositions forcancer vaccines that target cancer stem cells. Cancer stem cells areimportant in tumor maintenance, proliferation, and resistance tochemotherapy and radiation therapy. Thus, the new vaccines that targetcancer stem cells provide for greater therapeutic and/or prophylacticeffect, particularly in cancers that are resistant to conventionaltreatments.

In one aspect, this application provides methods of treating cancer(e.g., neural cancer) in a patient that include administering to thepatient a composition that includes antigen presenting cells (e.g.,dendritic cells) that present cancer stem cell antigens (e.g., neuralstem cell antigens).

In another aspect, this application provides methods of treating cancer(e.g., neural cancer) in a patient that include the steps of: obtaininga population of antigen presenting cells (e.g., dendritic cells);contacting the antigen presenting cells with a cancer stem cell antigencomposition (e.g., a neural cancer stem cell antigen composition) underconditions such that the antigen presenting cells present cancer stemcell antigens (e.g., neural cancer stem cell antigens); andadministering to a patient a composition that includes the antigenpresenting cells.

In a further aspect, this application provides methods of inducing orstimulating an immune response in a patient, and methods of generatingantibodies specific for cancer stem cell antigens, that includeadministering to the patient a composition that includes antigenpresenting cells (e.g., dendritic cells) that present cancer stem cellantigens (e.g., neural stem cell antigens).

In another aspect, this application provides methods of inducing orstimulating an immune response in a patient, and methods of generatingantibodies specific for cancer stem cell antigens, by obtaining apopulation of antigen presenting cells (e.g., dendritic cells);contacting the dendritic cells with a cancer stem cell antigencomposition (e.g., a neural cancer stem cell antigen composition) underconditions such that the antigen presenting cells present cancer stemcell antigens (e.g., neural cancer stem cell antigens); andadministering to a patient a composition that includes the antigenpresenting cells.

In a further aspect, this application provides methods of preparing acancer vaccine (e.g., a neural cancer vaccine), that include the stepsof obtaining a population of antigen presenting cells (e.g., dendriticcells), and contacting the antigen presenting cells with a cancer stemcell antigen composition (e.g., a neural cancer stem cell antigencomposition) under conditions such that the antigen presenting cellspresent cancer stem cell antigens (e.g., neural cancer stem cellantigens), thus preparing a cancer vaccine. In some embodiments, themethods further include administering the vaccine to a patient.

In another aspect, this application provides methods of preparing a cellvaccine for treating a cancer (e.g., a neural cancer) by obtainingmononuclear cells from a subject; culturing the mononuclear cells invitro under conditions in which mononuclear cells differentiate intoantigen presenting cells; isolating cancer stem cells (e.g., neuralcancer stem cells) from the same or different subject; preparing acancer stem cell antigen composition (e.g., a neural cancer stem cellantigen composition) from the cancer stem cells; and culturing theantigen presenting cells in the presence of the cancer stem cell antigencomposition, thus preparing a cell vaccine. In some embodiments, themethods further include administering the vaccine to a patient.

In some embodiments of any of the above aspects, the antigen presentingcells are autologous to the subject or patient. In some embodiments, theantigen presenting cells are allogeneic to the subject or patient.

In some embodiments of any of the above aspects, the cancer stem cellantigen composition is a lysate of cancer stem cells (e.g., neural stemcells). In other embodiments, the cancer stem cell antigen compositionis an acid eluate of cancer stem cells (e.g., neural cancer stem cells).

In some embodiments of any of the above aspects, the neural cancer stemcell antigen composition is obtained from a brain tumor (e.g., aglioma). In some embodiments of any of the above aspects, the cancerstem cells express CD133. In some embodiments of any of the aboveaspects, the cancer stem cell antigen composition includes one or moreisolated peptides of CD133, CD90, CD44, CXCR4, Nestin, Musashi-1 (Msi1),maternal embryonic leucine zipper kinase (MELK), GLI1, PTCH1, Bmi-1,phosphoserine phosphatase (PSP), Snail, OCT4, BCRP1, MGMT, Bcl-2, FLIP,BCL-XL, XIAP, cIAP1, cIAP2, NAIP, or survivin. In some embodiments, thepeptides are synthetic.

In another aspect, this application provides kits for preparing a cellvaccine for inducing an immune response or treating a cancer (e.g., abrain cancer) that include one or more isolated peptides of CD133, CD90,CD44, CXCR4, Nestin, Musashi-1 (Msi1), maternal embryonic leucine zipperkinase (MELK), GLI1, PTCH1, Bmi-1, phosphoserine phosphatase (PSP),Snail, OCT4, BCRP1, MGMT, Bcl-2, FLIP, BCL-XL, XIAP, cIAP1, cIAP2, NAIP,or survivin.

In a further aspect, this application provides compositions (e.g.,immunomodulatory compositions) that include antigen presenting cells(e.g., dendritic cells) that present cancer stem cell antigens (e.g.,neural cancer stem cell antigens). In some embodiments, the cancer stemcell antigens include peptides of one or more of CD133, CD90, CD44,CXCR4, Nestin, Musashi-1 (Msi1), maternal embryonic leucine zipperkinase (MELK), GLI1, PTCH1, Bmi-1, phosphoserine phosphatase (PSP),Snail, OCT4, BCRP1, MGMT, Bcl-2, FLIP, BCL-XL, XIAP, cIAP1, cIAP2, NAIP,or survivin. In some embodiments, the compositions are produced bymethods described herein.

In another aspect, this application provides for the use of compositions(e.g., immunomodulatory compositions) that include antigen presentingcells (e.g., dendritic cells) that present cancer stem cell antigens(e.g., neural cancer stem cell antigens) in the preparation of amedicament for modulating an immune response or treating cancer in asubject. In some embodiments, the cancer stem cell antigens includepeptides of one or more of CD133, CD90, CD44, CXCR4, Nestin, Musashi-1(Msi1), maternal embryonic leucine zipper kinase (MELK), GLI1, PTCH1,Bmi-1, phosphoserine phosphatase (PSP), Snail, OCT4, BCRP1, MGMT, Bcl-2,FLIP, BCL-XL, XIAP, cIAP1, cIAP2, NAIP, or survivin. In certainembodiments, the compositions are produced by methods described herein.

This application also provides immunogenic compositions that include, orencode cancer stem cell antigens, and methods of using the compositions.For example, preparations of cancer stem cell antigens, for use ascancer vaccines (e.g., peptide vaccines, DNA vaccines) are provided.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of a disease condition, inhibitingthe disease condition from worsening, improving symptoms of a diseasecondition, and prolonging a patient's life or life expectancy.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, breastcancer, colon cancer, lung cancer, prostate cancer, hepatocellularcancer, gastric cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer,and brain cancer; including, but not limited to, gliomas, glioblastomas,glioblastoma multiforme (GBM), oligodendrogliomas, primitiveneuroectodermal tumors low, mid and high grade astrocytomas, ependymomas(e.g., myxopapillary ependymoma papillary ependymoma, subependymoma,anaplastic ependymoma), oligodendrogliomas, medulloblastomas,meningiomas, pituitary adenomas, neuroblastomas, and craniopharyngiomas.

“Conditions” and “disease conditions,” as used herein may include, butare in no way limited to any form of neoplastic cell growth andproliferation, whether malignant or benign, pre-cancerous and cancerouscells and tissues; in particular, gliomas, glioblastomas, glioblastomamultiforme (GBM), oligodendrogliomas, primitive neuroectodermal tumors,low, mid and high grade astrocytomas, ependymomas (e.g., myxopapillaryependymoma papillary ependymoma, subependymoma, anaplastic ependymoma),oligodendrogliomas, medulloblastomas, meningiomas, pituitary adenomas,neuroblastomas, and craniopharyngiomas.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term. Theterms “patient” and “subject” are used herein interchangeably, and covermammals, including humans.

“Pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

“Stem-like” or “stem,” as used herein refers to cells that are able toself renew from a single clone, differentiate into terminal cell types,and be serially transplantable in immunodeficient animals.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder even if the treatment is ultimately unsuccessful. Those in needof treatment include those already diagnosed as having the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented. For example, in tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents or by the subject's own immune system.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference herein in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of an exemplary process for vaccination ofa patient with tumor antigen-pulsed dendritic cells. Monocytes areisolated from a patient and used to generate immature dendritic cells.After excision of a tumor or a portion of the tumor, brain tumorstem-like cells are isolated and used to prepare antigens forvaccination. The dendritic cells are pulsed with the antigens derivedfrom the brain tumor stem-like cells. The mature antigen-presentingdendritic cells are then used to vaccinate the patient, providing anincreased immune response against the tumor or residual tumor cells.

FIG. 2 is a schematic diagram depicting an exemplary method of isolationand generation of tumor neurospheres.

FIGS. 3A-3F are a set of histograms depicting FACS analysis ofneurospheres differentiated for 2 weeks. Neurospheres differentiated for2 weeks show (3B) 96% of cells positive for the astrocyte marker GFAP;(3C) 88% of cells positive for the NSC marker nestin; (3D) 29% of cellspositive for the neuronal marker MAP2; (3E) 0% of cells positive for theneuronal marker beta-tubulin III; (3F) 6% of cells positive for theoligodendrocyte marker myelin/oligodendrocyte. The samples were comparedto a negative control as shown (3A).

FIG. 4A is a graph depicting volume of tumors induced by neurospheres(n=4) and monolayer cells (n=4) at day 18. Tumors induced byneurospheres were significantly larger (p<0.02) than those from themonolayer group.

FIG. 4B is a line chart depicting survival of rats with tumors inducedby neurospheres (n=9) and monolayer cells (n=9). On average, animals inthe neurosphere group died at a significantly (p<0.02) earlier timepoint compared to those in the monolayer group.

FIGS. 5A-5E are a set of micrographs of tumors formed by implantation ofneurospheres. (5A) neurospheres formed high grade gliomas with necrosisas seen on H&E; (5B) large and well circumferential tumors grew asevidenced in non-stained sections; (5C) large and well circumferentialtumors grew as evidenced in sections stained for the nuclear markerDAPI; (5D) & (5E) comparison of non-tumor area (5D) and tumor area (5E)stained for reticulin reveals the high levels of reticulin formed intumor engulfed regions, showing the histological sarcomatous componentof the gliosarcoma. Scale bar 3000 μm (5A) Scale bar=1250 μm (5B, 5C);Scale bar=250 μm (5D, 5E).

FIGS. 6A-6B are line graphs depicting chemoresistance of neurospheresand monolayer cells to the indicated concentrations of carboplatin (6A)and Temodar (6B). Each value represents the mean of three independenttrials. Neurospheres were significantly more resistant (p<0.05) to bothchemotherapeutic agents as compared to monolayer cells.

FIG. 7 is a bar graph depicting proliferation rates of neurospheres andmonolayer cells in serum-containing media for 2 days. The WST-1proliferation assay was used to measure absorbance, which directlycorrelates to the cell number. The monolayer cells proliferated at asignificantly (p>0.05) greater rate than the neurosphere cells.

FIGS. 8A-8C are micrographs depicting primary culture of adult humanglioblastoma cells. (8A) Neurosphere-like tumor cells were found inglioblastoma primary cell culture in F— 12/DMEM 10% FBS medium. Scalebar=200 μm. (8B) Neurosphere derived from a single isolated CD133⁺ cellcultured in serum free medium with EGF/FGF. Scale bar=200 μm. (8C) CD133expression on a neurosphere derived from a single isolated CD133⁺ cellin serum free medium with EGF/FGF. Staining indicates CD133 expression.Scale bar=50 μm.

FIGS. 9A-9C are histograms depicting CD133 protein expression on primarycultured tumor cells. Tumor cells were cultured in F-12/DMEM 10% FBSmedium for 3-4 passages and stained with specific mAb to CD133 andisotype control-matched mAb. Results are given as the percentage ofCD133 positive cells in the total population. In the histograms, thethick lines represent staining with CD133 mAb, and the thin linesrepresent the isotype control-matched mAb. (9A) tumor cells from patientNo. 1049; (9B) tumor cells from patient No. 377; (9C) tumor cells frompatient No. 66.

FIGS. 10A-10D are line graphs depicting drug sensitivity of CD133positive cancer stem cells derived from patient No. 66. Tumor cells ofNo. 66 were cultured in FBS/F-12/DMEM medium for 4 passages. Both CD133positive and negative tumor cells were collected by FACS sorting. 1×10⁴cells/well were plated in 96-well plate and treated with the indicatedconcentrations of VP-16, Taxol, temozolomide, and carboplatin for 48hours in FBS/F-12/DMEM medium. * indicates p<0.05 compared to autologousCD133 negative cells. Data are representative of two independentexperiments. (10A) VP16; (10B) Taxol; (10C) temozolomide; (10D)carboplatin.

FIGS. 11A-11C are a set of line graphs depicting drug sensitivity ofCD133 positive cancer stem cells derived from patient Nos. 377 and 1049.Tumor cells of patient No. 377 and No. 1049 were cultured inFBS/F-12/DMEM medium for 3 passages. Both CD133 positive and negativetumor cells were collected by FACS sorting. 1×10⁴ cells/well were platedin 96-well plate and treated with the indicated concentrations ofcarboplatin (11A and 11C) or VP-16 (11B) for 48 hours in FBS/F-12/DMAEMmedium. * indicates p<0.05 compared to autologous CD133 negative cells.Data are representative of two independent experiments.

FIG. 12 is a bar graph depicting CD133 mRNA expression in primary (P)and recurrent (R) tumor tissue. Total RNA was extracted from bothprimary and recurrent tumor tissue derived from five patients, and CD133mRNA expression was measured by real-time qPCR. The relative CD133 mRNAlevel of recurrent tumor was presented as the fold increase compared tothat of autologous primary tumor tissue. Data are representative of twoindependent experiments.

FIG. 13 is a line graph depicting survival of 9L tumor-bearing ratsvaccinated with dendritic cells pulsed with antigens from 9L adherentcells (9L-AC), 9L daughter cells (9L-DC), 9L neurospheres (9L-NS), anddendritic cells alone (CONTROL). Kaplan-Meier survival curve showed thatthe 9L-DC group had significantly longer survival than the other groups(p 0.0015).

FIG. 14 is a bar graph depicting IFNγ production of splenocytesharvested from tumor bearing rats that were re-stimulated and by eitherre-exposure to a 9L tumor cell target (Target) or to no target (NOTarget). The re-stimulated splenocytes from NS vaccinated rats releasedhigh level of IFNγ when re-exposed to NS, whereas splenocytes from ratsvaccinated with AC or DtC had no detectable IFNγ in response tore-exposure to AC or DC.

FIGS. 15A and 15B are a pair of micrographs of tumor slices from ratsvaccinated with dendritic cells pulsed with daughter cells (17A) andneurospheres (17B) and stained with anti-CD4 antibody. Greaterinfiltration of CD4⁺ cells was observed in the tumors of rats vaccinatedwith neurosphere-pulsed dendritic cells. (Magnification ×40).

FIG. 16 is a structural diagram of CD133 depicting extracellular,intracellular, and transmembrane regions of CD133.

FIGS. 17A and 17B are cell plots and histograms depicting expression ofisotype control (17A) and MHC class I expression (17B) in human cancerstem cells.

FIGS. 18A and 18B are cell plots and histograms depicting expression ofisotype control (18A) and MHC class I expression (18B) in human neuralstem cells.

DETAILED DESCRIPTION

The present application describes compositions useful as vaccines fortreating cancer (e.g., neural cancers, e.g., gliomas) that includedendritic cells pulsed with antigens obtained from cancer stem cells(e.g., neurospheres); methods of producing vaccines that includedendritic cells pulsed with antigens obtained from cancer stem cells;methods of treating cancer with vaccines that include dendritic cellspulsed with antigens obtained from cancer stem cells; and kits fortreating cancer that include dendritic cells pulsed with antigensobtained from cancer stem cells.

Greater tumor infiltration of cytotoxic T cells was observed in animalsvaccinated against cancer stem cell antigens, and a stronger responseagainst tumor cells was observed in T cells isolated from animalsvaccinated with cancer stem cell antigens, than in responses in whichantigens were not prepared from stem cell enriched cell populations.Cancer stem cells obtained from brain tumors were capable ofself-renewal and proliferation, and could recapitulate the tumor wheninjected into rats. Isolated cancer stem cells formed more aggressivetumors as compared to differentiated tumor cells in vitro, and thecancer stem cells showed a higher resistance to chemotherapeutic agents.Similarly, CD133-positive cancer stem cells were obtained from humantumors. These cells were similarly resistant to chemotherapeutic agentsand CD133-positive cells were found at a higher level in patients inwhom tumors had recurred following resection.

Vaccination Methods

Described herein are methods of vaccinating a subject, e.g., to treatcancer (e.g., neural cancer, e.g., gliomas) with antigen-presentingcells (“APC”), e.g., dendritic cells (“DC”), that include antigens fromcancer stem cells, e.g., presented on the surface of theantigen-presenting cells. Dendritic cells (e.g., autologous orallogeneic dendritic cells) are contacted with cancer stem cell antigensas a cell lysate, acid elution, cell extract, partially purifiedantigens, purified antigens, isolated antigens, partially purifiedpeptides, purified peptides, isolated peptides, synthetic peptides, or acombination of two or more of the above. The antigen-presenting cellsare then administered to a subject in need of cancer vaccination (e.g.,a subject diagnosed with or at risk for cancer) to treat the cancer.FIG. 1 is a schematic diagram of an exemplary process for vaccination ofa patient with cancer stem cell antigen-pulsed dendritic cells.

Cancer Stem Cells

The “cancer stem cell” hypothesis proposes that only a small portion ofa tumor is represented by the “cancer stem cell,” which allows the tumorto proliferate and self renew, and eventually differentiate into thephenotypically diverse and heterogeneous tumor cell population (Bjerkviget al., Nat. Rev. Cancer, 5:899-904, 2005). Cancer stem cells can beisolated from any type of cancers, e.g., leukemias (Bonnet and Dick,Nat. Med., 3:730-737, 1997), breast cancers (Al-Hajj et al., Proc. Natl.Acad. Sci. USA, 100:3983-88, 2003), colon cancers (O'Brien et al.,Nature, 445:106-110, 2007), and brain cancers (Singh et al., Nature,432:396-401, 2004; Hemmati et al., Proc. Natl. Acad. Sci. USA,100:15178-83, 2003; Singh et al., Cancer Res., 63:5821-28, 2003; Sanaiet al., N. Engl. J. Med., 353:811-822, 2005; Tunici et al., Mol. Cancer,3:25, 2004). Cancer stem cells are characterized by their ability toself-renew and proliferate, and recapitulate through differentiation thetumor from which it is isolated. Additionally, neural cancer stem cellsform clonally derived neurospheres in culture.

Cancer stem cells can be isolated by dissociating tumor cells andculturing them under conditions that promote proliferation of stem cells(e.g., conditions that inhibit differentiation of the stem cells).Methods and conditions for isolating stem cells are known in the art.Exemplary methods and conditions can be found in U.S. Pat. No.5,589,376, U.S. Pat. No. 5,643,741, U.S. Pat. No. 5,650,299, U.S. Pat.No. 5,824,489, U.S. Pat. No. 5,849,553, U.S. Pat. No. 5,928,947, U.S.Pat. No. 5,981,708, U.S. Pat. No. 6,337,184, U.S. Pat. No. 6,645,763,U.S. Pat. No. 6,800,790, U.S. Pat. No. 6,875,607, U.S. Pat. No.6,984,522, U.S. Pat. No. 7,109,032, U.S. Pat. No. 7,115,267, and U.S.Pat. No. 7,115,360.

Additionally, cancer stem cells can be identified or isolated (e.g.,isolated from non-stem tumor cells) on the basis of expression (e.g.,nucleic acid or protein expression) of molecular markers, e.g.,molecular markers described in the U.S. patents referred to in the aboveparagraph. Exemplary molecular markers include CD133, Bmi-1, Notch,Sonic hedgehog, and Wnt. Additionally, exemplary molecular markers ofneural cancer stem cells include CD90, CD44, CXCR4, Nestin, Musashi-1(Msi1), maternal embryonic leucine zipper kinase (MELK), GLI1, PTCH1,Bmi-1, phosphoserine phosphatase (PSP), Snail, OCT4, BCRP1, MGMT, Bcl-2,FLIP, BCL-XL, XIAP, cIAP1, cIAP2, NAIP, and survivin.

Isolation or identification of cancer stein cells can be performed bystandard means, e.g., cell sorting (e.g., fluorescence activated cellsorting (FACS) or magnetic cell sorting (MACS)).

Antigens

Antigenic peptides useful for loading DCs for vaccination are peptidesthat stimulate a T cell mediated immune response (e.g., a cytotoxic Tcell response) by presentation to T cells on MHC molecules. Usefulantigenic peptides and proteins include those derived from cancer steincells (e.g., neural cancer stem cells, CD133⁺ tumor cells, orneurospheres derived from tumors). In some embodiments, the cancer stemcell antigens are presented as a lysate of the cancer stem cells. Inother embodiments, the cancer stem cell antigens are obtained by acidelution of peptides presented on MHC molecules of the cancer stem cells.In an exemplary method, cancer stem cells are washed with an isotonicsolution (e.g., Hank's buffered saline solution) to remove mediacomponents. The cells are then treated with acid (e.g., citratephosphate buffer, pH 3.2) to dissociate peptides from surface MHCs, andthe cells removed from the solution containing the soluble peptides. Theacid-eluted cancer stem cell peptide antigens can be further purified(e.g., on a C18 column) and frozen for storage prior to use.

Specific antigens that can be used in the methods described hereininclude portions of the amino acid sequences of CD133, CD90, CD44,CXCR4, Nestin, Musashi-1 (Msi1), maternal embryonic leucine zipperkinase (MELK), GLI1, PTCH1, Bmi-1, phosphoserine phosphatase (PSP),Snail, OCT4, BCRP1, MGMT, Bcl-2, FLIP, BCL-XL, XIAP, cIAP1, cIAP2. NAIP,and survivin that bind to MHC molecules and are presented to T cells.Peptides that bind to MHC class I molecules are generally 8-10 aminoacids in length. Peptides that bind to MHC class II molecules aregenerally 13 amino acids or longer (e.g., 13-17 amino acids long).

CD133 is a 120 kDa, five-transmembrane domain glycoprotein expressed onneural and hematopoietic stem and progenitor cells (Yin et al., Blood,90:5002-12, 1997). Table 1 provides an amino acid sequence of CD133(also available in GenBank under accession number NP_(—)006008.1,GI:5174387). The structure of CD133 includes an extracellularN-terminus, two short intracellular loops, two large extracellularloops, and an intracellular C-terminus (FIG. 18). Exemplary CD133 T cellepitopes include 8-10 or 13-20 contiguous amino acid residues of aminoacid residues 325-350 of SEQ ID NO:53. An alternately spliced version ofCD133 is described in Yu et al., J. Biol. Chem., 277:20711-16, 2002.

CD90 is a cell surface glycoprotein found on T cells and neurons. Table1 provides an amino acid sequence of CD90 (also available in GenBankunder accession number NP_(—)006279.2, GI:19923362).

CD44 is a cell surface glycoprotein that may be involved in matrixadhesion. Table 1 provides an amino acid sequence of CD44 (alsoavailable in GenBank under accession number NP_(—)000601.3,GI:48255935). Several isoforms of CD44 are produced, primarily byalternative splicing (see Marhaba et al., J. Mol. Histol., 35:211-31,2004; Zoeller, Cancer Immunol. Immunother., 53:567-79, 2004).

CXCR4 is a chemokine receptor that has been found to be expressed inbreast cancers (Muller et al., Nature, 410:50-56, 2001). Table 1provides an amino acid sequence of CXCR4 (also available in GenBankunder accession number NP_(—)001008540.1, GI:56790927). An alternativespliced variant is available in GenBank under accession numberNP_(—)003458.1, GI:4503175.

Nestin is an intermediate filament protein expressed in neuralprogenitor cells (Dahlstrand et al., J. Cell Sci., 103:589-597, 1992).Table 1 provides an amino acid sequence of Nestin (also available inGenBank under accession number NP_(—)006608.1, GI:38176300).

Musashi-1 (Msi1) is an RNA-binding protein expressed in neuralprogenitor cells (Good et al., Genomics, 52:382-384, 1998; Siddall etal., Proc. Nat. Acad. Sci. USA, 103:84-8407, 2006; Okano et al., Exp.Cell Res., 306:349-356, 2005). Table 1 provides an amino acid sequenceof Musashi-1 (also available in GenBank under accession numberNP_(—)002433.1, GI:4505255).

Maternal embryonic leucine zipper kinase (MELK) is a protein kinaseexpressed in multiple cancers (Gray et al. Cancer Res., 65:9751-61,2005). Table 1 provides an amino acid sequence of MELK (also availablein GenBank under accession number NP_(—)055606.1, GI:7661974).

GLI1 is a zinc-finger transcription factor upregulated in cancers,including gliomas (Kinzler et al., Science, 236:70-73, 1987; Kinzler etal., Nature, 332:371-374, 1988; Kasper et al., Eur. J. Cancer,42:437-445, 2006). Table 1 provides an amino acid sequence of GLI1 (alsoavailable in GenBank under accession number NP_(—)005260.1, GI:4885279).

PTCH1 is a transmembrane protein that is believed to function as a tumorsuppressor (Katoh et al., Cancer Biol. Ther., 4:1050-54, 2005). Table 1provides an amino acid sequence of PTCH1 (also available in GenBankunder accession number NP_(—)000255.2, GI:134254446). Five isoforms ofPTCH1 are produced by alternative splicing (Nagao et al., Genomics,85:462-71, 2005).

Bmi-1 is a polycomb ring finger protein involved in proliferation ofprogenitor cells (Lessard et al., Nature, 423:255-260, 2003; Park etal., Nature, 423:302-305, 2003; Molofsky et al., Nature, 425:962-967,2003). Bmi-1 can play a role in the malignant transformation of the HOXA9/MEIS-induced murine leukemia model (Lessard et al., Nature,423:255-260, 2003) as well as in tumors of neural origin (van Lohuizenet al., Nature, 353:353-355, 1991). Table 1 provides an amino acidsequence of Bmi-1 (also available in GenBank under accession numberNP_(—)005171.4, GI:27883842). Exemplary Bmi-1 T cell epitopes includeTLQDIVYKL (SEQ ID NO:86), CLPSPSTPV (SEQ ID NO:87), VRYLETSKY (SEQ IDNO:88), KRYLRCPAA (SEQ ID NO:89), YEEEPLKDY (SEQ ID NO:90), andKEEVNDKRY (SEQ ID NO:91) (Steele et al., Br. J. Cancer 95:1202-11,2006).

Phosphoserine phosphatase (PSP) is an enzyme that catalyzes thehydrolysis of O-phosphoserine. Table 1 provides an amino acid sequenceof PSP (also available in GenBank under accession number NP_(—)004568.2,GI:46249388).

Snail is a zinc-finger transcription factor and anti-apoptotic protein(Vega et al., Genes Dev., 18:1131-1143, 2004). Table 1 provides an aminoacid sequence of Snail (also available in GenBank under accession numberNP_(—)005976.2, GI:18765741).

OCT4 is a POU homeodomain-containing transcription factor expressed inpluripotent cells (Nichols et al., Cell, 95:379-391, 1998). Table 1provides an amino acid sequence of OCT4 (also available in GenBank underaccession number NP_(—)002692.2, GI:42560248). An alternate isoform ofOCT4 is available in GenBank under accession number NP 976034.3,GI:116235491.

BCRP1 is an ATP-binding cassette (ABC) transporter protein involved inmultidrug resistance of tumors (Doyle et al., Proc. Nat. Acad. Sci. USA,95:1566570, 1998). Table 1 provides an amino acid sequence of BCRP1(also available in GenBank under accession number NP_(—)004818.2,GI:62526033).

MGMT is an O-6-methylguanine-DNA methyltransferase DNA-mismatch repairprotein that can provide resistance to some methylating andchloroethylating agents, such as temozolomide (Rabik et al., CancerTreat. Rev., 32:261-276, 2006; Cai et al., Cancer Res., 65:3319-27,2005). Table 1 provides an amino acid sequence of MGMT (also availablein GenBank under accession number NP_(—)002403.1, GI:4505177).

BCL-2 is a mitochondrial anti-apoptotic protein correlated withchemotherapy resistant cancers and decreased overall survival (Campos etal., Blood, 81:3091-3096, 1993). Table 1 provides an amino acid sequenceof BCL-2 (also available in GenBank under accession numberNP_(—)000624.2, GI:72198189). An alternatively spliced isoform of BCL-2is available in GenBank under accession number NP_(—)000648.2,GI:72198346.

FLIP is an anti-apoptotic protein (Irmler et al., Nature, 388:190-195,1997). Table 1 provides an amino acid sequence of FLIP (also availablein GenBank under accession number NP_(—)003870.3, GI:21361769).

BCL-XL is an anti-apoptotic protein related to BCL-2, which may beinvolved in chemoresistance (Boise et al., Cell, 74:597-608, 1993;Andreeff et al., Leukemia, 13:1881-92, 1999). Table 1 provides an aminoacid sequence of BCL-XL (also available in GenBank under accessionnumber NP_(—)612815.1, GI:20336335). Exemplary T cell epitopes of BCL-XLinclude Bcl-xL118-126 (TAYQSFEQV; SEQ ID NO:92), Bcl-xL173-182(YLNDHLEPWI; SEQ ID NO:93), and Bcl-xL169-178 (WMATYLNDHL; SEQ ID NO:94)(Andersen et al., J. Immunol., 175:2709-14, 2005).

XIAP is a member of the inhibitor of apoptosis protein (IAP) family(Deveraux et al., Nature, 388:300-304, 1997). Table 1 provides an aminoacid sequence of XIAP (also available in GenBank under accession numberNP_(—)001158.2, GI:32528299).

cIAP1 is a member of the IAP family of apoptosis inhibitors (Rothe etal., Cell, 83: 1243-1252, 1995; Liston et al., Nature, 379:349-353,1996). Table 1 provides an amino acid sequence of cIAP1 (also availablein GenBank under accession number NP_(—)001157.1, GI:4502141).

cIAP2 is a member of the IAP family of apoptosis inhibitors (Liston etal., Nature, 379:349-353, 1996). Table 1 provides an amino acid sequenceof cIAP2 (also available in GenBank under accession numberNP_(—)031490.1, GI:6680696).

NAIP is a member of the IAP family of apoptosis inhibitors (Roy et al.,Cell, 80:167-178, 1995). Table 1 provides an amino acid sequence of NAIP(also available in GenBank under accession number NP_(—)004527.2,GI:19393878). An alternatively spliced isoform of NAIP is available inGenBank under accession number NP_(—)075043.1, GI:119393876.

Survivin is a member of the IAP family of apoptosis inhibitors (Li etal., Nature, 396:580-584, 1998). Table 1 provides an amino acid sequenceof NAIP (also available in GenBank under accession numberNP_(—)001159.2, GI:59859878). Alternatively spliced isoforms of survivinhave been identified (see Wheatley et al., Int. Rev. Cytol., 247:35-88,2005; Noton et al., J. Biol. Chem., 281:1286-95, 2006; and Taubert etal., Oncogene, 24:5258-61, 2005). Sequences of exemplary alternativeisoforms are available in GenBank under accession numbersNP_(—)001012270.1, GI:59859880, and NP_(—)001012271.1, GI:59859882.Exemplary T cell epitopes of survivin include Sur20-28 (STFKNWPFL; SEQID NO:95), Sur96-104 (LTLGEFLKL; SEQ ID NO:96), Sur133-141 (RAIEQLAAM;SEQ ID NO:97), and Sur126-135 (ETAKKVRRAI; SEQ ID NO:98) (Bachinsky etal., Cancer Immun., 5:6, 2005). Other exemplary T cell epitopes ofsurvivin include Sur92-101 (QFEELTLGEF; SEQ ID NO:99), Sur54-62(LAQCFFCFK; SEQ ID NO:100), Sur112-120 (KIAKETNNK; SEQ ID NO:101),Sur53-62 (DLAQCFFCFK; SEQ ID NO:102), Sur112-121 (KIAKETNNKK; SEQ IDNO:103), Sur18-28 (RISTFKNWPFL; SEQ ID NO:104), Sur86-96 (FLSVKKQFEEL;SEQ ID NO:105), and the modified peptides Sur92T2 (QTEELTLGEF; SEQ IDNO:67), Sur93T2 (FTELTLGEF; SEQ ID NO:68), Sur93S2 (FSELTLGEF; SEQ IDNO:69), Sur38Y9 (MAEAGFIHY; SEQ ID NO:70), Sur47Y10 (PTENEPDLAY; SEQ IDNO:71), Sur5K9 (TLPPAWQPK; SEQ ID NO:72), Sur54L2 (LLQCFFCFK; SEQ IDNO:73), and Sur18K10 (RISTFKNWPK; SEQ ID NO:74) (Reker et al., CancerBiol. Ther., 3:173-179, 2004). Other exemplary T cell epitopes ofsurvivin include ELTLGEFLKL (SEQ ID NO:75) and TLPPAWQPFL (SEQ ID NO:76)(Schmitz et al., Cancer Res. 60:4845-4849, 2000). Additional survivinepitopes are described in Siegel et al., Br. J. Haematol., 122:911-914,2003.

TABLE 1 Sequences of Human Antigens Antigen Amino acid sequence(SEQ ID NO: 53) CD133MALVLGSLLLLGLCGNSFSGGQPSSTDAPKAWNYELPATNYETQDSHKAGPIGILFELVHIFLYVVQPRDFPEDTLRKFLQKAYESKIDYDKPETVILGLKIVYYEAGIILCCVLGLLFIILMPLVGYFFCMCRCCNKCGGEMHQRQKENGPFLRKCFAISLLVICIIISIGIFYGFVANHQVRTRIKRSRKLADSNFKDLRTLLNETPEQIKYILAQYNTTKDKAFTDLNSINSVLGGGILDRLRPNIIPVLDEIKSMATAIKETKEALENMNSTLKSLHQQSTQLSSSLTSVKTSLRSSLNDPLCLVHPSSETCNSIRLSLSQLNSNPELRQLPPVDAELDNVNNVLRTDLDGLVQQGYQSLNDIPDRVQRQTTTVVAGIKRVLNSIGSDIDNVTQRLPIQDILSAFSVYVNNTESYIHRNLPTLEEYDSYWWLGGLVICSLLTLIVIFYYLGLLCGVCGYDRHATPTTRGCVSNTGGVFLMVGVGLSFLFCWILMIIVVLTFVFGANVEKLICEPYTSKELFRVLDTPYLLNEDWEYYLSGKLFNKSKMKLTFEQVYSDCKKNRGTYGTLHLQNSFNISEHLNINEHTGSISSELESLKVNLNIFLLGAAGRKNLQDFAACGIDRMNYDSYLAQTGKSPAGVNLLSFAYDLEAKANSLPPGNLRNSLKRDAQTIKTIHQQRVLPIEQSLSTLYQSVKILQRTGNGLLERVTRILASLDFAQNFITNNTSSVIIEETKKYGRTIIGYFEHYLQWIEFSISEKVASCKPVATALDTAVDVFLCSYIIDPLNLFWFGIGKATVFLLPALIFAVKLAKYYRRMDSEDVYDDVETIPMKNMENGNNGYHKDHVYGIHNPVMTSPSQH (SEQ ID NO: 54) CD90MNLAISIALLLTVLQVSRGQKVTSLTACLVDQSLRLDCRHENTSSSPIQYEFSLTRETKKHVLFGTVGVPEHTYRSRTNFTSKYNMKVLYLSAFTSKDEGTYTCALHHSGHSPPISSQNVTVLRDKLVKCEGISLLAQNTSWLLLLLLSLSLLQATDFMSL (SEQ ID NO: 55) CD44MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSISRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQKGEYRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTDRIPATTLMSTSATATETATKRQETWDWFSWLFLPSESKNHLHTTTQMAGTSSNTISAGWEPNEENEDERDRHLSFSGSGIDDDEDFISSTISTTPRAFDHTKQNQDWTQWNPSHSNPEVLLQTTTRMTDVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEETPHSTSTIQATPSSTTEETATQKEQWFGNRWHEGYRQTPKEDSHSTTGTAAASAHTSHPMQGRTTPSPEDSSWTDFFNPISHPMGRGHQAGRRMDMDSSHSITLQPTANPNTGLVEDLDRTGPLSMTTQQSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRRDPNHSEGSTTLLEGYTSHYPHTKESRTFIPVTSAKTGSFGVTAVTVGDSNSNVNRSLSGDQDTFHPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPEWLIILASLLALALILAVCIAVNSRRRCGQKKKLVINSGNGAVEDRKPSGLNGEASKSQEMVHLVNKESSETPDQFMTADETRN LQNVDMKIGV(SEQ ID NO: 56) CXCR4MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS  (SEQ ID NO: 57)Nestin MEGCMGEESFQMWELNRRLEAYLARVKALEEQNELLSAELGGLRAQSADTSWRAHADDELAALRALVDQRWREKHAAEVARDNLAEELEGVAGRCQQLRLARERTTEEVARNRRAVEAEKCARAWLSSQVAELERELEALRVAHEEERVGLNAQAACAPRCPAPPRGPPAPAPEVEELARRLGEAWRGAVRGYQERVAHMETSLGQARERLGRAVQGAREGRLELQQLQAERGGLLERRAALEQRLEGRWQERLRATEKFQLAVEALEQEKQGLQSQIAQVLEGRQQLAHLKMSLSLEVATYRTLLEAENSRLQTPGGGSKTSLSFQDPKLELQFPRTPEGRRLGSLLPVLSPTSLPSPLPATLETPVPAFLKNQEFLQARTPTLASTPIPPTPQAPSPAVDAEIRAQDAPLSLLQTQGGRKQAPEPLRAEARVAIPASVLPGPEEPGGQRQEASTGQSPEDHASLAPPLSPDHSSLEAKDGESGGSRVFSICRGEGEGQIWGLVEKETAIEGKVVSSLQQEIWEEEDLNRKEIQDSQVPLEKETLKSLGEEIQESLKTLENQSHETLERENQECPRSLEEDLETLKSLEKENKELLKDVEVVRPLEKEAVGQLKPTGKEDTQTLQSLQKENQELMKSLEGNLETFLFPGTENQELVSSLQENLESLTALEKENQEPLRSPEVGDEEALRPLTKENQEPLRSLEDENKEAFRSLEKENQEPLKTLEEEDQSIVRPLETENHKSLRSLEEQDQETLRTLEKETQQRRRSLGEQDQMTLRPPEKVDLEPLKSLDQEIARPLENENQEFLKSLKEESVEAVKSLETEILESLKSAGQENLETLKSPETQAPLWTPEEINQGAMNPLEKEIQEPLESVEVNQETFRLLEEENQESLRSLGAWNLENLRSPEEVDKESQRNLEEEENLGKGEYQESLRSLEEEGQELPQSADVQRWEDTVEKDQELAQESPPGMAGVENEDEAELNLREQDGFTGKEEVVEQGELNATEEVWIPGEGHPESPEPKEQRGLVEGASVKGGAEGLQDPEGQSQQVGAPGLQAPQGLPEAIEPLVEDDVAPGGDQASPEVMLGSEPAMGESAAGAEPGPGQGVGGLGDPGHLTREEVMEPPLEEESLEAKRVQGLEGPRKDLEEAGGLGTEFSELPGKSRDPWEPPREGREESEAEAPRGAEEAFPAETLGHTGSDAPSPWPLGSEEAEEDVPPVLVSPSPTYTPILEDAPGPQPQAEGSQEASWGVQGRAEALGKVESEQEELGSGEIPEGPQEEGEESREESEEDELGETLPDSTPLGFYLRSPTSPRWDPTGEQRPPPQGETGKEGWDPAVLASEGLEAPPSEKEEGEEGEEECGRDSDLSEEFEDLGTEAPFLPGVPGEVAEPLGQVPQLLLDPAAWDRDGESDGFADEEESGEEGEEDQEEGREPGAGRWGPGSSVGSLQALSSSQRGEFLESDSVSVSVPWDDSLRGAVAGAPKTALETESQDSAEPSGSEEESDPVSLEREDKVPGPLEIPSGMEDAGPGADIIGVNGQGPNLEGKSQHVNGGVMNGLEQSEEVGQGMPLVSEGDRGSPFQEEEGSALKTSWAGAPVHLGQGQFLKFTQREGDRESWSSGED (SEQ ID NO: 58) Msi1METDAPQPGLASPDSPHDPCKMFIGGLSWQTTQEGLREYFGQFGEVKECLVMRDPLTKRSRGFGFVTFMDQAGVDKVLAQSRHELDSKTIDPKVAFPRRAQPKMVTRTKKIFVGGLSVNTTVEDVKQYFEQFGKVDDAMLMFDKTTNRHRGFGFVTFESEDIVEKVCEIHFHEINNKMVECKKAQPKEVMSPTGSARGRSRVMPYGMDAFMLGIGMLGYPGFQATTYASRSYTGLAPGYTYQFPEFRVERTPLPSAPVLPELTAIPLTAYGPMAAAAAAAAVVRGTGSHPWTMAPPPGSTPSRTGGFLGTTSPGPMAELYGAANQDSGVSSYISAASPAPSTGFGHSLGGPLIATAFTNGYH(SEQ ID NO: 59) MELKMKDYDELLKYYELHETIGTGGFAKVKLACHILTGEMVAIKIMDKNTLGSDLPRIKTEIEALKNLRHQHICQLYHVLETANKIFMVLEYCPGGELFDYIISQDRLSEEETRVVFRQIVSAVAYVHSQGYAHRDLKPENLLFDEYHKLKLIDFGLCAKPKGNKDYHLQTCCGSLAYAAPELIQGKSYLGSEADVWSMGILLYVLMCGFLPFDDDNVMALYKKIMRGKYDVPKWLSPSSILLLQQMLQVDPKKRISMKNLLNHPWIMQDYNYPVEWQSKNPFIHLDDDCVTELSVHHRNNRQTMEDLISLWQYDHLTATYLLLLAKKARGKPVRLRLSSFSCGQASATPFTDIKSNNWSLEDVTASDKNYVAGLIDYDWCEDDLSTGAATPRTSQFTKYWTESNGVESKSLTPALCRTPANKLKNKENVYTPKSAVKNEEYFMFPEPKTPVNKNQHKREILTTPNRYTTPSKARNQCLKETPIKIPVNSTGTDKLMTGVISPERRCRSVELDLNQAHMEETPKRKGAKVFGSLERGLDKVITVLTRSKRKGSARDGPRRLKLHYNVTTTRLVNPDQLLNEIMSILPKKHVDFVQKGYTLKCQTQSDFGKVTMQFELEVCQLQKPDVVGIRRQRLKGDAWVYKRLVEDILSSCKV (SEQ ID NO: 60) GLI1MFNSMTPPPISSYGEPCCLRPLPSQGAPSVGTEGLSGPPFCHQANLMSGPHSYGPARETNSCTEGPLFSSPRSAVKLTKKRALSISPLSDASLDLQTVIRTSPSSLVAFINSRCTSPGGSYGHLSIGTMSPSLGFPAQMNHQKGPSPSFGVQPCGPHDSARGGMIPHPQSRGPFPTCQLKSELDMLVGKCREEPLEGDMSSPNSTGIQDPLLGMLDGREDLEREEKREPESVYETDCRWDGCSQEFDSQEQLVHHINSEHIHGERKEFVCHWGGCSRELRPFKAQYMLVVHMRRHTGEKPHKCTFEGCRKSYSRLENLKTHLRSHTGEKPYMCEHEGCSKAFSNASDRAKHQNRTHSNEKPYVCKLPGCTKRYTDPSSLRKHVKTVHGPDAHVTKRHRGDGPLPRAPSISTVEPKREREGGPIREESRLTVPEGAMKPQPSPGAQSSCSSDHSPAGSAANTDSGVEMTGNAGGSTEDLSSLDEGPCIAGTGLSTLRRLENLRLDQLHQLRPIGTRGLKLPSLSHTGTTVSRRVGPPVSLERRSSSSSSISSAYTVSRRSSLASPFPPGSPPENGASSLPGLMPAQHYLLRARYASARGGGTSPTAASSLDRIGGLPMPPWRSRAEYPGYNPNAGVTRRASDPAQAADRPAPARVQRFKSLGCVHTPPTVAGGGQNFDPYLPTSVYSPQPPSITENAAMDARGLQEEPEVGTSMVGSGLNPYMDFPPTDTLGYGGPEGAAAEPYGARGPGSLPLGPGPPTNYGPNPCPQQASYPDPTQETWGEFPSHSGLYPGPKALGGTYSQCPRLEHYGQVQVKPEQGCPVGSDSTGLAPCLNAHPSEGPPHPQPLFSHYPQPSPPQYLQSGPYTQPPPDYLPSEPRPCLDFDSPTHSTGQLKAQLVCNYVQSQQELLWEGGGREDAPAQEPSYQSPKFLGGSQVSPSRAKAPVNTYGPGFGPNLPNHKSGSYPTPSPCHENFVVGANRASHRAAAPPRLLPPLPTCYGPLKVGGTNPSCGHPEVGRLGGGPALYPPPEGQVCNPLDSLDLDNTQLDFVAILDEPQGLSPPPSHDQRGSSGHTPPPSGPPNMAVGNMSVLLRSLPGE TEFLNSSA(SEQ ID NO: 61) PTCH1MASAGNAAEPQDRGGGGSGCIGAPGRPAGGGRRRRTGGLRRAAAPDRDYLHRPSYCDAAFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKAANLETNVEELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQHLDSALQASRVHVYMYNRQWKLEHLCYKSGELITETGYMDQIIEYLYPCLIITPLDCFWEGAKLQSGTAYLLGKPPLRWTNFDPLEFLEELKKINYQVDSWEEMLNKAEVGHGYMDRPCLNPADPDCPATAPNKNSTKPLDMALVLNGGCHGLSRKYMHWQEELIVGGTVKNSTGKLVSAHALQTMFQLMTPKQMYEHFKGYEYVSHINWNEDKAAAILEAWQRTYVEVVHQSVAQNSTQKVLSFTTTTLDDILKSFSDVSVIRVASGYLLMLAYACLTMLRWDCSKSQGAVGLAGVLLVALSVAAGLGLCSLIGISFNAATTQVLPFLALGVGVDDVFLLAHAFSETGQNKRIPFEDRTGECLKRTGASVALTSISNVTAFFMAALIPIPALRAFSLQAAVVVVFNFAMVLLIFPAILSMDLYRREDRRLDIFCCFTSPCVSRVIQVEPQAYTDTHDNTRYSPPPPYSSHSFAHETQITMQSTVQLRTEYDPHTHVYYTTAEPRSEISVQPVTVTQDTLSCQSPESTSSTRDLLSQFSDSSLHCLEPPCTKWTLSSFAEKHYAPFLLKPKAKVVVIFLFLGLLGVSLYGTTRVRDGLDLTDIVPRETREYDFIAAQFKYFSFYNMYIVTQKADYPNIQHLLYDLHRSFSNVKYVMLEENKQLPKMWLHYFRDWLQGLQDAFDSDWETGKIMPNNYKNGSDDGVLAYKLLVQTGSRDKPIDISQLTKQRLVDADGIINPSAFYIYLTAWVSNDPVAYAASQANIRPHRPEWVHDKADYMPETRLRIPAAEPIEYAQFPFYLNGLRDTSDFVEAIEKVRTICSNYTSLGLSSYPNGYPFLFWEQYIGLRHWLLLFISVVLACTFLVCAVFLLNPWTAGIIVMVLALMTVELFGMMGLIGIKLSAVPVVILIASVGIGVEFTVHVALAFLTAIGDKNRRAVLALEHMFAPVLDGAVSTLLGVLMLAGSEFDFIVRYFFAVLAILTILGVLNGLVLLPVLLSFFGPYPEVSPANGLNRLPTPSPEPPPSVVRFAMPPGHTHSGSDSSDSEYSSQTTVSGLSEELRHYEAQQGAGGPAHQVIVEATENPVFAHSTVVHPESRHHPPSNPRQQPHLDSGSLPPGRQGQQPRRDPPREGLWPPPYRPRRDAFEISTEGHSGPSNRARWGPRGARSHNPRNPASTAMGSSVPGYCQPITTVTASASVTVAVHPPPVPGPGRNPRGGLCPGYPETDHGLFEDPHVPFHVRCERRDSKVEVIELQDVECEERPRGSSSN (SEQ ID NO: 62) Bmi-1MHRTTRIKITELNPHLMCVLCGGYFIDATTIIECLHSFCKTCIVRYLETSKYCPICDVQVHKTRPLLNIRSDKTLQDIVYKLVPGLFKNEMKRRRDFYAAHPSADAANGSNEDRGEVADEDKRIITDDEIISLSIEFFDQNRLDRKVNKDKEKSKEEVNDKRYLRCPAAMTVMHLRKFLRSKMDIPNTFQIDVMYEEEPLKDYYTLMDIAYIYTWRRNGPLPLKYRVRPTCKRMKISHQRDGLTNAGELESDSGSDKANSPAGGIPSTSSCLPSPSTPVQSPHPQFPHISSTMNGTSNSPSGNHQSSFANRPRKSSVNGSSATSSG (SEQ ID NO: 63) PSPMVSHSELRKLFYSADAVCFDVDSTVIREEGIDELAKICGVEDAVSEMTRRAMGGAVPFKAALTERLALIQPSREQVQRLIAEQPPHLTPGIRELVSRLQERNVQVFLISGGFRSIVEHVASKLNIPATNVFANRLKFYFNGEYAGFDETQPTAESGGKGKVIKLLKEKFHFKKIIMIGDGATDMEACPPADAFIGFGGNVIRQQVKDNAKWYITDFVELLGELEE (SEQ ID NO: 64) SnailMPRSFLVRKPSDPNRKPNYSELQDSNPEFTFQQPYDQAHLLAAIPPPEILNPTASLPMLIWDSVLAPQAQPIAWASLRLQESPRVAELTSLSDEDSGKGSQPPSPPSPAPSSFSSTSVSSLEAEAYAAFPGLGQVPKQLAQLSEAKDLQARKAFNCKYCNKEYLSLGALKMHIRSHTLPCVCGTCGKAFSRPWLLQGHVRTHTGEKPFSCPHCSRAFADRSNLRAHLQTHSDVKKYQCQACARTFSRMSLLHKHQESGCSGCPR (SEQ ID NO: 65) OCT4MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN (SEQ ID NO: 66)BCRP1 MSSSNVEVFIPVSQGNTNGFPATASNDLKAFTEGAVLSFHNICYRVKLKSGFLPCRKPVEKEILSNINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPSGLSGDVLINGAPRPANFKCNSGYVVQDDVVMGTLTVRENLQFSAALRLATTMTNHEKNERINRVIQELGLDKVADSKVGTQFIRGVSGGERKRTSIGMELITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTIIFSIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAYNNPADFFLDIINGDSTAVALNREEDFKATEIIEPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEKKKKITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQIIVTVVLGLVIGAIYFGLKNDSTGIQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYISGYYRVSSYFLGKLLSDLLPMRMLPSIIFTCIVYFMLGLKPKADAFFVMMFTLMMVAYSASSMALAIAAGQSVVSVATLLMTICFVFMMIFSGLLVNLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATGNNPCNYATCTGEEYLVKQGIDLSPWGLWKNHVALACMIVIFLTIAYLKLLFLKKYS (SEQ ID NO: 77) MGMTMDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEVPAPAAVLGGPEPLMQCTAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPKAARAVGGAMRGNPVPILIPCHRVVCSSGAVGNYSGGLAVKEWLLAHEGHRLGKPGLGGSSGLAGAWLKGAGATSGSPPAGRN (SEQ ID NO: 78) BCL-2MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK (SEQ ID NO: 79)FLIP MSAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAELLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIFLMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKYKQSVQGAGTSYRNVLQAAIQKSLKDPSNNFRLHNGRSKEQRLKEQLGAQQEPVKKSIQESEAFLPQSIPEERYKMKSKPLGICLIIDCIGNETELLRDTFTSLGYEVQKFLHLSMHGISQILGQFACMPEHRDYDSFVCVLVSRGGSQSVYGVDQTHSGLPLHHIRRMFMGDSCPYLAGKPKMFFIQNYVVSEGQLENSSLLEVDGPAMKNVEFKAQKRGLCTVHREADFFWSLCTADMSLLEQSHSSPSLYLQCLSQKLRQERKRPLLDLHIELNGYMYDWNSRVSAKEKYYVWLQHTLRKKLILSYT (SEQ ID NO: 80)BCL-XL MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK (SEQ ID NO: 81) XIAPMTFNSFEGSKTCVPADINKEEEFVEEFNRLKTFANFPSGSPVSASTLARAGFLYTGEGDTVRCFSCHAAVDRWQYGDSAVGRHRKVSPNCRFINGFYLENSATQSTNSGIQNGQYKVENYLGSRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNWPDYAHLTPRELASAGLYYTGIGDQVQCFCCGGKLKNWEPCDRAWSEHRRHFPNCFFVLGRNLNIRSESDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGFYALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTHSLEECLVRTTEKTPSLTRRIDDTIFQNPMVQEAIRMGFSFKDIKKIMEEKIQISGSNYKSLEVLVADLVNAQKDSMQDESSQTSLQKEISTEEQLRRLQEEKLCKICMDRNIAIVFVPCGHLVTCKQCAEAVDKCPMCYTVI TFKQKIFMS(SEQ ID NO: 82) cIAP1MHKTASQRLFPGPSYQNIKSIMEDSTILSDWTNSNKQKMKYDFSCELYRMSTYSTFPAGVPVSERSLARAGFYYTGVNDKVKCFCCGLMLDNWKLGDSPIQKHKQLYPSCSFIQNLVSASLGSTSKNTSPMRNSFAHSLSPTLEHSSLFSGSYSSLSPNPLNSRAVEDISSSRTNPYSYAMSTEEARFLTYHMWPLTFLSPSELARAGFYYIGPGDRVACFACGGKLSNWEPKDDAMSEHRRHFPNCPFLENSLETLRFSISNLSMQTHAARMRTFMYWPSSVPVQPEQLASAGFYYVGRNDDVKCFCCDGGLRCWESGDDPWVEHAKWFPRCEFLIRMKGQEFVDEIQGRYPHLLEQLLSTSDTTGEENADPPIIHFGPGESSSEDAVMMNTPVVKSALEMGFNRDLVKQTVQSKILTTGENYKTVNDIVSALLNAEDEKREEEKEKQAEEMASDDLSLIRKNRMALFQQLTCVLPILDNLLKANVINKQEHDIIKQKTQIPLQARELIDTILVKGNAAANIFKNCLKEIDSTLYKNLFVDKNMKYIPTEDVSGLSLEEQLRRLQEERTCKVCMDKEVSVVFIPCGHLVVCQECAPSLRKCPICRGIIK GTVRTFLS(SEQ ID NO: 83) cIAP2MVQDSAFLAKLMKSADTFELKYDFSCELYRLSTYSAFPRGVPVSERSLARAGFYYTGANDKVKCFCCGLMLDNWKQGDSPMEKHRKLYPSCNFVQTLNPANSLEASPRPSLPSTAMSTMPLSFASSENTGYFSGSYSSFPSDPVNFRANQDCPALSTSPYHFAMNTEKARLLTYETWPLSFLSPAKLAKAGFYYIGPGDRVACFACDGKLSNWERKDDAMSEHQRHFPSCPFLKDLGQSASRYTVSNLSMQTHAARIRTFSNWPSSALVHSQELASAGFYYTGHSDDVKCFCCDGGLRCWESGDDPWVEHAKWFPRCEYLLRIKGQEFVSQVQAGYPHLLEQLLSTSDSPEDENADAAIVHFGPGESSEDVVMMSTPVVKAALEMGFSRSLVRQTVQRQILATGENYRTVSDLVIGLLDAEDEMREEQMEQAAEEEESDDLALIRKNKMVLFQHLTCVTPMLYCLLSARAITEQECNAVKQKPHTLQASTLIDTVLAKGNTAATSFRNSLREIDPALYRDIFVQQDIRSLPTDDIAALPMEEQLRKLQEERMCKVCMDREVSIVFIPCGHLVVCKDCAPSLRKCPICRGTIKGTVRTFLS (SEQ ID NO: 84) NAIPMATQQKASDERISQFDHNLLPELSALLGLDAVQLAKELEEEEQKERAKMQKGYNSQMRSEAKRLKTFVTYEPYSSWIPQEMAAAGFYFTGVKSGIQCFCCSLILFGAGLTRLPIEDHKRFHPDCGFLLNKDVGNIAKYDIRVKNLKSRLRGGKMRYQEEEARLASFRNWPFYVQGISPCVLSEAGFVFTGKQDTVQCFSCGGCLGNWEEGDDPWKEHAKWFPKCEFLRSKKSSEEITQYIQSYKGFVDITGEHFVNSWVQRELPMASAYCNDSIFAYEELRLDSFKDWPRESAVGVAALAKAGLFYTGIKDIVQCFSCGGCLEKWQEGDDPLDDHTRCFPNCPFLQNMKSSAEVTPDLQSRGELCELLETTSESNLEDSIAVGPIVPEMAQGEAQWFQEAKNLNEQLRAAYTSASFRHMSLLDISSDLATDHLLGCDLSIASKHISKPVQEPLVLPEVFGNLNSVMCVEGEAGSGKTVLLKKIAFLWASGCCPLLNRFQLVFYLSLSSTRPDEGLASIICDQLLEKEGSVTEMCVRNIIQQLKNQVLFLLDDYKEICSIPQVIGKLIQKNHLSRTCLLIAVRTNRARDIRRYLETILEIKAFPFYNTVCILRKLFSHNMTRLRKFMVYFGKNQSLQKIQKTPLFVAAICAHWFQYPFDPSFDDVAVFKSYMERLSLRNKATAEILKATVSSCGELALKGFFSCCFEFNDDDLAEAGVDEDEDLTMCLMSKFTAQRLRPFYRFLSPAFQEFLAGMRLIELLDSDRQEHQDLGLYHLKQINSPMMTVSAYNNFLNYVSSLPSTKAGPKIVSHLLHLVDNKESLENISENDDYLKHQPEISLQMQLLRGLWQICPQAYFSMVSEHLLVLALKTAYQSNTVAACSPFVLQFLQGRTLTLGALNLQYFFDHPESLSLLRSIHFPIRGNKTSPRAHFSVLETCFDKSQVPTIDQDYASAFEPMNEWERNLAEKEDNVKSYMDMQRRASPDLSTGYWKLSPKQYKIPCLEVDVNDIDVVGQDMLEILMTVFSASQRIELHLNHSRGFIESIRPALELSKASVTKCSISKLELSAAEQELLLTLPSLESLEVSGTIQSQDQIFPNLDKFLCLKELSVDLEGNINVFSVIPEEFPNFHHMEKLLIQISAEYDPSKLVKLIQNSPNLHVFHLKCNFFSDFGSLMTMLVSCKKLTEIKFSDSFFQAVPFVASLPNFISLKILNLEGQQFPDEETSEKFAYILGSLSNLEELILPTGDGIYRVAKLIIQQCQQLHCLRVLSFFKTLNDDSVVEIAKVAISGGFQKLENLKLSINHKITEEGYRNFFQALDNMPNLQELDISRHFTECIKAQATTVKSLSQCVLRLPRLIRLNMLSWLLDADDIALLNVMKERHPQSKYLTILQKWILPFSPIIQK(SEQ ID NO: 85) SurvivinMGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAEKVRRAIEQLAAMD

T cell epitopes can be identified by a number of different methods.Naturally processed MHC epitopes can be identified by massspectrophotometric analysis of peptides eluted from antigen-loaded APC(e.g., APC that have taken up antigen, or that have been engineered toproduce the protein intracellularly). After incubation at 37° C., cellsare lysed in detergent and the MHC protein is purified (e.g., byaffinity chromatography). Treatment of the purified MHC with a suitablechemical medium (e.g., under acidic conditions) results in the elutionof peptides from the MHC. This pool of peptides is separated and theprofile compared with peptides from control APC treated in the same way.The peaks unique to the protein expressing/fed cells are analyzed (forexample by mass spectrometry) and the peptide fragments identified. Thisprotocol identifies peptides generated from a particular antigen byantigen processing.

Alternatively, epitopes can be identified by screening a syntheticlibrary of peptides that overlap and span the length of the antigen inan in vitro assay. For example, peptides that are 9 amino acids inlength and which overlap by 5 amino acids may be used. The peptides aretested in an antigen presentation system that includes antigenpresenting cells and T cells. T cell activation in the presence of APCspresenting the peptide can be measured (e.g., by measuring T cellproliferation or cytokine production) and compared to controls, todetermine whether a particular epitope is recognized by the T cells.

T cell epitopes can be predicted in silico, e.g., using the methodsdescribed in Parker et al., J. Immunol., 152:163, 1994 and Rammensee etal., Immunogenet., 50:213-219, 1999.

Antigenic peptides can be obtained by chemical synthesis using acommercially available automated peptide synthesizer. Synthetic peptidescan be precipitated and further purified, for example by highperformance liquid chromatography (HPLC). Alternatively, isolatedpeptides can be obtained by purification and/or recombinant methodsusing host cell and vector expression systems.

Preparation of Antigen Presenting Cells

Antigen presenting cells (APC), such as DCs, suitable for administrationto subjects (e.g., glioma patients) can be isolated or obtained from anytissue in which such cells are found, or may be otherwise cultured andprovided using standard techniques. Methods of preparing antigenpresenting cells are well-known to those of skill in the art. Maturedendritic cells are typically identified as having the following cellsurface marker phenotype: MAC3⁻, CD80⁺, CD86⁺, CD40^(low), CD54⁺, MHCClass I and MHC Class II, and are capable of FITC-dextran uptake.

APCs (e.g., DCs) can be found, by way of example, in the bone marrow orPBMCs of a mammal, in the spleen of a mammal or in the skin of a mammal(i.e., Langerhan's cells, which possess certain qualities similar tothat of DC, may be found in the skin). For instance, bone marrow can beharvested from a mammal and cultured in a medium that promotes thegrowth of DCs. GM-CSF, IL-4 and/or other cytokines (e.g., TNF-α), growthfactors and supplements may be included in this medium.

After a suitable amount of time in culture in medium containingappropriate cytokines (e.g., time suitable to expand and differentiatethe DCs into mature DCs, e.g., 2, 4, 6, 8, 10, 12, or 15 days), clustersof DCs are cultured in the presence of a sufficient number of antigensof interest (e.g., in the presence of cancer stem cell lysate, acideluted peptides of cancer stem cells, peptides of CD133, CD90, CD44,CXCR4, Nestin, Musashi-1 (Msi1), maternal embryonic leucine zipperkinase (MELK), GLI1, PTCH1, Bmi-1, phosphoserine phosphatase (PSP),Snail, OCT4, BCRP1, MGMT, Bcl-2, FLIP, BCL-XL, XIAP, cIAP1, cIAP2, NAIP,or survivin, or a combination of two or more of the above antigens) andharvested for use in a cancer vaccine. For example, peptide antigens canbe added to the culture medium at a concentration of about 1.0 to 50,e.g., 5, 10, 15, 20, or 30 μg/ml (per antigen).

Alternately, or in combination, antigens can be transgenically expressedin DCs, e.g., by transfection of nucleic acids encoding one or more ofthe antigens or portions of antigens.

In one exemplary method, APCs are isolated from a subject (e.g., ahuman) according to the following exemplary procedure. Mononuclear cellsare isolated from blood using leukapheresis (e.g., using a COBE SpectraApheresis System). The mononuclear cells are allowed to become adherentby incubation in tissue culture flasks for 2 hours at 37° C. Nonadherentcells are removed by washing. Adherent cells are cultured in mediumsupplemented with granulocyte macrophage colony stimulating factor(GM-CSF) and interleukin-4 (IL-4) for five days. On day five, TNF-α isadded to the culture medium for another 3-4 days. On day 8 or 9, cellsare harvested and washed, and incubated with peptide antigens for 16-20hours on a tissue rotator. Peptide antigens are added to the cultures ata concentration of ˜10 μg/ml (per antigen).

Various other methods can be used to isolate the APCs, as would berecognized by one of skill in the art. DCs occur in low numbers in alltissues in which they reside, making isolation and enrichment of DCs arequirement. Any of a number of procedures entailing repetitive densitygradient separation, fluorescence activated cell sorting techniques,positive selection, negative selection or a combination thereof areroutinely used to obtain enriched populations or isolated DCs. Guidanceon such methods for isolating DCs can be found in O'Doherty et al., J.Exp. Med., 178:1067-78, 1993; Young and Steinman, J. Exp. Med.,171:1315-32, 1990; Freudenthal and Steinman, Proc. Nat. Acad. Sci. USA,57:7698-7702, 1990; Macatonia et al., 67:285-289, 1989; Markowicz andEngleman, J. Clin. Invest., 85:955-961, 1990; Mehta-Damani et al., J.Immunol., 153:996-1003, 1994; and Thomas et al., J. Immunol.,151:6840-6852, 1993. One method for isolating DCs from human peripheralblood is described in U.S. Pat. No. 5,643,786. Methods of producing DCsfrom embryonic stem cells are described in U.S. Pat. No. 7,247,480.

Administration of Cancer Vaccines

The APC-based cancer vaccine may be delivered to a recipient by anysuitable delivery route, which can include injection, infusion,inoculation, direct surgical delivery, or any combination thereof. Insome embodiments, the cancer vaccine is administered to a human in thedeltoid region or axillary region. In some embodiments, the vaccine isadministered to a subject locally to the site of a tumor, within thetumor, or to an area from which a tumor has been surgically resected.

An appropriate carrier for administering the cells may be selected byone of skill in the art by routine techniques. For example, thepharmaceutical carrier can be a buffered saline solution, e.g., cellculture media.

The quantity of APC appropriate for administration to a patient as acancer vaccine to effect the methods of the present invention and themost convenient route of such administration may be based upon a varietyof factors, as may the formulation of the vaccine itself. Some of thesefactors include the physical characteristics of the patient (e.g., age,weight, and sex), the physical characteristics of the tumor (e.g.,location, size, rate of growth, and accessibility), and the extent towhich other therapeutic methodologies (e.g., chemotherapy, and beamradiation therapy) are being implemented in connection with an overalltreatment regimen. Notwithstanding the variety of factors one shouldconsider in implementing the methods of the present invention to treat adisease condition, a mammal can be administered with from about 10⁵ toabout 10⁹ APC (e.g., 10⁷ APC) in from about 0.05 mL to about 5 mLsolution (e.g., saline) in a single administration. Additionaladministrations can be carried out, depending upon the above-describedand other factors, such as the severity of tumor pathology. In oneembodiment, from about one to about five administrations of about 10⁶APC is performed at two-week intervals.

DC vaccination can be accompanied by other treatments. For example, apatient receiving DC vaccination may also be receiving chemotherapy,radiation, and/or surgical therapy concurrently. Methods of treatingcancer using DC vaccination in conjunction with chemotherapy aredescribed in Wheeler et al., U.S. Pat. Pub. No. 2007/0020297. In someembodiments, a patient receiving DC vaccination has already receivedchemotherapy, radiation, and/or surgical treatment for the cancer. Inone embodiment, a patient receiving DC vaccination is treated with aCOX-2 inhibitor, as described in Yu and Akasaki, WO 2005/037995.

Pharmaceutical Compositions

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of the inventive vaccinecomprising dendritic cells pulsed with cancer stem cell antigens asdescribed herein. “Pharmaceutically acceptable excipient” means anexcipient that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and desirable, and includes excipientsthat are acceptable for veterinary use as well as for humanpharmaceutical use. Such excipients may be solid, liquid, semisolid, or,in the case of an aerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, transmucosal, transdermal, or parenteral. “Parenteral”refers to a route of administration that is generally associated withinjection, including intraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid tiller, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Kits

Kits to treat cancer are also contemplated. The kits are useful forpracticing the inventive method of treating cancer with a vaccinecomprising dendritic cells pulsed with cancer stem cell antigens asdescribed herein. The kit is an assemblage of materials or components,including at least one of the inventive compositions. Thus, in someembodiments the kit contains a composition including a vaccinecomprising dendritic cells pulsed with cancer stem cell antigens asdescribed herein.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of treating a particular cancer. In oneembodiment, the kit is configured for the purpose of treating braintumors. In one particular embodiment, the brain tumor is a glioma. Inanother embodiment, the brain tumor is GBM. In one embodiment, the kitis configured particularly for the purpose of treating mammaliansubjects. In another embodiment, the kit is configured particularly forthe purpose of treating human subjects. In further embodiments, the kitis configured for veterinary applications, treating subjects such as,but not limited to, farm animals, domestic animals, and laboratoryanimals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as induction of an immune response against a tumor, to treat acancer. For example, the instructions may comprise instructions toadminister a vaccine comprising dendritic cells pulsed with cancer stemcell antigens to the patient.

Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, or other usefulparaphernalia as will be readily recognized by those of skill in theart.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in cancer treatments or in vaccinations. As used herein, theterm “package” refers to a suitable solid matrix or material such asglass, plastic, paper, foil, and the like, capable of holding theindividual kit components. Thus, for example, a package can be a glassvial used to contain suitable quantities of an inventive compositioncontaining for example, a vaccine comprising dendritic cells pulsed withcancer stem cell antigens as described herein. The packaging materialgenerally has an external label which indicates the contents and/orpurpose of the kit and/or its components.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 The 9L Gliosarcoma Cell Line Contains Self-Renewing Cells

To determine if a population of self-renewing stem cells exist withinthe phenotypically heterogeneous 9L gliosarcoma tumor, the cells weregrown as monolayers in the presence of 10% FBS and subsequently grewthem in serum free media containing mitogens. A schematic diagram ofculturing of tumor stem cells is presented in FIG. 2. 9L gliosarcomaswere resuspended in Dulbecco's modified Eagle's medium/F-12 mediumcontaining 10% fetal bovine serum (FBS) and plated at a density of 1×10⁶live cells per 75 cm² flask. The cells attached and grew as monolayersand were passaged upon confluency. Spheres were derived by placing the9L gliosarcomas cells grown as monolayers into a defined serum-free NSCmedium (Reynolds et al., J. Neurosci., 12: 4565-74, 1992; Reynolds etal., Science, 255:1707-10, 1992) consisting of Dulbecco's modifiedEagle's medium/F-12 medium supplemented with 20 ng/mL of both epidermalgrowth factor (EGF; Peprotech, Rocky Hill, N.J.) and basic fibroblastgrowth factor (bFGF; Peprotech, Rocky Hill, N.J.). Cells were fed every2 days by adding fresh NSC media supplemented with growth factors. Afterprimary spheres formed and reached 10-200 cells per sphere, the cellswere harvested, dissociated into single cells using trypsin and EDTA(GIBCO BRL) and mechanical pipetting, strained through a cell strainer,and plated at a clonal density of 1,000 cells/mL inneurosphere-conditioned medium to generate clonally derived subspheres(Geschwind et al., Neuron, 29:325-339, 2001; Groszer et al., Science,294: 2186-89, 2001). The cells were fed every 2 days by adding fresh NSCmedia supplemented with mitogens. Cells and subsequent spheres wereobserved daily for 18 days, and passaged into fresh media. Subspheresranging from approximately 15 cells to 40 cells were evident after 18days and displayed the self-renewing and proliferative capacity of the9L spheres.

Subtle differences were observed in surface adhesion properties betweenthe two cell populations when using different coating solutions. It wasfound that poly-L-lysine proved less effective in binding toneurospheres than did laminin. Even after 2 days, only a small portionof the cells adhered (˜5%). However, when neurospheres were allowed toadhere to chamber slides coated with laminin, nearly all the cells(˜70%) adhered within the first 3 hours. As a consequence, poly-L-lysinewas not used as an adhesive substrate in further studies. A similartrend was observed when comparing adhesion of monolayers to chamberslides coated with either poly-L-lysine or laminin—cellular morphologywas more apparent in monolayers grown on laminin coated chamber slideswithin a 6 hour time period. Furthermore, a distinct morphologicalcharacteristic was apparent in the differentiated neurospheres, wherearms of differentiated cells originating from one neurosphere homed toarms formed by another nearby neurosphere (FIG. 8A). These examples ofextracellular adhesion and cellular homing can be used to distinguishcancer stem-like cells (CSLCs) from non-CSLCs within a tumor.

Example 2 9L Neurospheres Express NSC Markers and can Generate BothNeuronal and Glial Cells in Culture

To determine the expression of markers for stem cells, neurospheres wereimmunostained for NSC markers nestin and Sox2. Neurospheres were alsostained for the lineage markers for astrocytes, GFAP, neurons,beta-tubulin III and MAP2, and oligodendrocytes, myelin/oligodendrocyte.Cells in the outer region of the neurosphere labeled for nestin, whilecells throughout the neurosphere were labeled for Sox2. A large numberof cells within the tumor spheres were also found to be positive for thelineage marker GFAP, while relatively few cells expressed the neuronallineage markers β-tubulin III, MAP2, and myelin/oligodendrocyte.

To test whether spheres have multipotent capabilities and produceprogenies of different lineages, spheres were seeded into chamber slides(Lab-TekII, Nalge Nunc International) for differentiation assay. Thecells were grown for 14 days in medium devoid of growth factors bFGF andEGF but permissive for differentiation, and processed forimmunocytochemistry as described below. The medium included Dulbecco'smodified Eagle's medium/F-12 medium containing 10% fetal bovine serum(FBS).

To examine the expression of NSC markers and lineage markers, immuncyto-and immunohistochemical staining was performed. For staining ofdifferentiated spheres, spheres and 9L monolayers, cells growing inchamber slides were fixed with 4% paraformaldehyde for 15 minutes at 4°C., treated with 5% NHS (normal horse serum)/0.1% Triton-X, and thenstained with the following antibodies: rabbit anti-nestin (1:200,Chemicon), rabbit anti-Sox2 (1:1,000, Chemicon), rabbit anti-MAP2(1:1,000, Sigma), mouse anti-β-tubulin III (1:200, Chemicon), rabbitanti-GFAP (1′:1000, Chemicon), mouse anti-myelin/oligodendrocyte(1:1,000, Chemicon). The primary antibodies were detected with Cy3 orFITC-conjugated anti-mouse or anti-rabbit IgG antibody (1:200, JacksonImmuno Research). The cells were counterstained with4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) to identifyall nuclei. The stained sections were examined and photographed using aQED cell scanner program and Nikon Eclipse TE2000-E microscope, andanalyzed using Image J (NIH). For immunostaining of spheres, the sphereswhere allowed to adhere to precoated (with laminin) chamber slides for 3hours before fixation, while monolayers were allowed to adhere overnightin non-fixed chamber slides.

Cells stained positive for the NSC markers nestin and Sox2, as well asthe lineage markers for astrocytes, GFAP, neurons, MAP2, andoligodendrocytes, myelin/oligodendrocyte. However, no cells were labeledwith the neuronal marker beta-tubulin III. FACS analysis showed similarresults to those derived via immunocytochemistry, which showeddifferentiated cells labeled for nestin (88%; FIG. 3C), Sox2 (24%), GFAP(96%; FIG. 3B), MAP2 (29%; FIG. 3D), and myelin/oligodendrocyte (6%;FIG. 3F), but not for β-tubulin III (0%; FIG. 3E). For FACS analysis,cells were fixed with 4% paraformaldehyde for 15 minutes at 4° C.,treated with 5% NHS (normal horse serum)/0.1% Triton™-X, and thenstained with the following antibodies: rabbit anti-nestin (1:1,000,Chemicon), rabbit anti-Sox2 (1:1,000, Chemicon), rabbit anti-MAP2(1:1,000, Sigma), mouse anti-β-tubulin III (1:500, Chemicon), rabbitanti-GFAP (1:1000, Chemicon), and mouse anti-myelin/oligodendrocyte(1:1,000, Chemicon). The primary antibodies were detected withFITC-conjugated anti-mouse or anti-rabbit IgG antibody (1:200, JacksonImmuno Research) using 10⁴ cells in a FACSVantage™ fluorescenceactivated cell sorter (Becton Dickinson). These results indicate thatthese cancer stem-like cells (CSLCs), unlike normal stem cells,differentiate into aberrant cells that are positive for multipledifferentiation markers, notably GFAP and MAP2. Such a dual nature isapparent in the majority of cells differentiated from neurospheres.Furthermore, many cells still remained highly positive for nestin andSox2. The expression pattern of the differentiated progeny was similarin profile to that of primary cultured tumor cells from which thespheres had originally been isolated and predominantly differentiatedinto GFAP and MAP2 positive cells that recapitulated the parental tumorphenotype. Additionally, the level of labeling for the NSC markersnestin and Sox2 still remained high, even after 14 days ofdifferentiation. The staining pattern for the monolayer population wassimilar to neurospheres differentiated for 14 days, except for theexpression of Sox2, which was greater in neurospheres differentiated for14 days. These results reveal that 9L spheres are multipotent for thethree neural cell types and differentiate into cells found in theoriginal tumor from which they were obtained.

Example 3 The Aggressiveness of 9L Cells In Vivo is Reliant on theNeurosphere Cancer Stem-Like Cells

To determine if 9L cells grown as monolayer or neurospheres differ intheir ability to grow as a tumor after implantation 5,000 cells fromboth populations of cells were injected into rats and survival and tumorvolume were assayed.

Fisher F344 rats 6-8 weeks old (Harlan Sprague-Dawley, Indianapolis,Ind.) were anesthetized with i.p. ketamine and xylazine, andstereotactically implanted in the right striatum (from bregma in mm:anterior-posterior +1 mm; medial-lateral −3 mm; dorsal-ventral −5 mm)with either isolated 9L sphere cells containing the luciferase gene(5,000 cells) or non-sphere-forming monolayer cells containing theluciferase gene (5,000 cells) in 4 μL of 1.2% methylcellulose/PBS(Rehemtulla et al., Neoplasia, 2:491-495, 2000). Rats were portioned toeither the tumor volume group (n=10) or the survival group (n=18), withcontrol rats (n=6) receiving 4 μL of 1.2% methylcellulose/PBS only.

Because half of each group consisted of either animals implanted withspheres or monolayers, tumor aggression could be determined. Animals inthe tumor volume group were sacrificed 18 days after tumor implantation.Tumor volume was assessed by using the formula for an ellipsoid,(length×width×height)/2 (Advani et al., Cancer Res., 59:2055-58, 1999),with the height and the width of the tumor being approximately equalbecause of the well-defined circumference of the tumors generated by the9L gliosarcoma (see Sibenaller et al., Neurosurg. Focus, 19:E1, 2005). Asignificantly (P<0.02) greater tumor volume was observed in the ratsimplanted with neurosphere cells as compared to rats implanted withmonolayer cells (FIG. 4A).

Animals in the survival group were followed for survival and euthanizedvia CO₂ asphyxiation when terminal neurological signs developed (e.g.,inability to access food, water, seizure activity, weakness, andparalysis) or if animals exceeded a survival period of 40 days.Following euthanization, brains were harvested and frozen in2-methylbutane (Sigma), cooled to −20° C., and stored at −80° C. untilsectioning. For H&E staining, 20 μm coronal brain sections describedabove were mounted on slide and stained with Harris hematoxylin for 2minutes and then counterstained with alcoholic eosin. Reticulin stainswere performed on 12 μm coronal brain sections.

Similar to the results seen in the tumor size groups, rats implantedwith neurospheres on average had shorter survival times than ratsimplanted with monolayer cells (FIG. 4B). Six rats in the neurospheregroup had large tumors that lead to terminal neurological symptoms,whereas 3 showed evidence of small tumors after 40 days of survival asseen by H&E staining of brain sections. In contrast, only 4 rats hadtumors large enough to create terminal neurological symptoms in themonolayer group, whereas 5 showed evidence of small tumor or engraftmentafter 40 days of survival as determined by H&E staining. Furthermore,the rats in the monolayer group which developed terminal neurologicalsigns of tumor did so at a later time (36 days compared to 29 days inthe neurosphere group), which was significant when analyzed using theKaplan-Meier test (P<0.02).

To determine the establishment of tumor in vivo, 150 mg/kg of theluciferase substrate D-luciferin (Biosynth, International, Inc.,Naperville, Ill.) was administered i.p. to animals in the survivalgroup, and luciferase scans were taken 15 minutes later. Luciferasescans generated at 14 days post implantation demonstrated a greaterproportion of animals with tumor burden in the neurosphere groupcompared to the monolayer group, even though a higher expression levelof luciferase in vitro was observed in monolayer cells as compared toneurosphere cells.

To determine if the 91, neurospheres could recapitulate the 9L sarcomain vivo, histological analysis of the tumors was performed. Thetumor-cell-implanted rat brains were cut with a cryostat into 20 μmcoronal sections and fixed in 4% paraformaldehyde, washed with PBS, andair dried. To characterize the brain tissue by immunohistochemistry,free-floating sections were blocked with 5% NHS (normal horse serum) for30 minutes at room temperature and then stained with the followingantibodies: rabbit anti-nestin (1:200, Chemicon), rabbit anti-Sox2(1:1,000, Chemicon), rabbit anti-MAP2 (1:1,000, Sigma), mouseanti-β-tubulin III (1:200, Chemicon), rabbit anti-GFAP (1:1000,Chemicon), mouse anti-myelin/oligodendrocyte (1:1000; Chemicon). Theprimary antibodies were detected as in Example 1. The cells werecounterstained with 4′,6-diamidino-2-phenylindole (DAPI; VectorLaboratories) to identify all nuclei. The stained sections were examinedand photographed using the Zeiss Axiovision 3.1 program in conjunctionwith Zeiss Axioskop™ 2 microscope, and analyzed using Image J (NIH).

Tumors from the neurosphere cell population were large andwell-circumferential, and showed cells positive for the NSC markernestin, as well as cells positive for the lineage markers GFAP,β-tubulin III, and myelin/oligodendrocytes. Most of the labeling withinthe tumor volume was directed against GFAP, and a lesser degree oflabeling was observed for β-tubulin III, myelin/oligodendrocyte, andnestin. A significant portion (>75%) of the tumor volume stainedpositive for reticulin, consistent with a sarcomatous component.Additionally, H&E staining revealed a high grade glioma with necrosisconsistent with a glioblastoma, displaying the dual nature of thegliosarcoma. Neurospheres formed high grade gliomas with necrosis asseen on H&E (FIG. 5A). The tumors were large and well circumferential asevidenced in non-stained sections (FIG. 5B) and stained for the nuclearmarker DAPI (FIG. 5D). A comparison of non-tumor area (FIG. 5D) withtumor area (FIG. 5E) stained for reticulin revealed high levels ofreticulin in tumor engulfed regions, showing the histologicalsarcomatous component of the gliosarcoma. The staining patterns suggestthat 9L neurospheres recapitulated the original tumor by differentiatinginto both neural and glial lineages in vivo.

Example 4 Proliferation Rate and Drug Sensitivity of 9L Neurospheres

To compare the differential proliferation rates and resistance tochemotherapeutic agents, 2,000 healthy 9L sphere and monolayer cellswere exposed to either Dulbecco's modified Eagle's medium/F-12 mediumcontaining 10% fetal bovine serum (FBS), or 100 μM stock solution ofTemozolamide or Carboplatin dissolved in PBS at concentrations of 1,000μM, 500 μM, 250 μM, and 125 μM for 2 days. The viability of the cellswas scored by measurement of the absorption of formazan dye (the amountof formazan dye formed directly correlates to the number ofmetabolically active cells) using the cellular proliferation assay WST-1(Roche Molecular Biochemicals, Mannheim, Germany). Formazan was measuredwith the use of a microplate reader (Tecan) and spectrophotometer set ata wavelength of 440 nm and a reference wavelength of 890 nm. Cellularviability was determined by exposing cells to WST-1 for 4 hours, andcalculating the percentage of viable cells. Proliferation was alsoassessed by using manual cell counting after 7 days in culture, with aninitial cellular concentration of 100,000 cells/mL in a 25 mm² flask.

The neurospheres demonstrated significantly (P<0.05) greater resistanceto temozolamide (FIG. 6B) and carboplatin (FIG. 6A) when compared to the9L cells grown as a monolayer under the same conditions. When the cellswere not treated with the chemotherapeutic agents and simply grown inmedium, there was a significantly (P<0.05) greater increase in cellnumber in the monolayer group by a factor of 1.48 when using the WST-1proliferation assay when the cells were not treated with thechemotherapeutic agents. The results of the proliferation assays aredepicted in FIG. 7, which is a bar graph that shows a greater increasein cells in the monolayer group. A similar trend in the untreated cellswas also observed using the manual cell count method, which showed agreater increase in monolayers by a factor of 1.35. This exampledemonstrates that neurosphere cells have a greater resistance tochemotherapeutic agents.

Example 5 Isolation of Human CD133-Positive Cancer Stem Cells

Glioblastoma specimens were obtained from patients (with informedconsent) via the Brain Tumor Registry and were reviewed and released bya pathologist in the operating room. Independent pathologists classifiedthe tumors by type and grade in accordance with the WHO histologicalgrading of central nervous system tumors. IRB certified techniciansprocessed the glioma tissues under sterile conditions in a laminar flowhood. Tumor cells were cultured in the following complete medium: Ham'sF-12/DMEM with high glucose (Irvine scientific, Santa Ana, Calif.), 10mM HEPES (Invitrogen, Carlsbad, Calif.), 0.1 mg/ml Gentamicin(Invitrogen) and 10% heat-inactivated FBS (Irvine Scientific, Santa Ana,Calif.). The cultured cells were maintained for 3-4 passages. Floatingneurosphere-like cells were obtained that were capable of forming newspheres in medium containing FBS for 3-4 passages.

Three adult glioblastoma primary tumor cell lines (Nos. 1049, 377, and66) were derived by the above method and analyzed by FACS for CD133expression. Tumor cells were collected and stained with anti-CD133antibody (mouse monoclonal IgG1; 1:10; Milteny Biotec) or IgG1 isotypecontrol antibody (BD Pharmingen, San Diego, Calif.). After PE-anti-mouseIgG1 (BD Pharmingen) staining for 30 minutes, CD133 staining wasanalyzed by flow cytometry using a FACSCalibur™ fluorescence activatedcell sorter (Becton Dickinson, San Jose, Calif.). CD133 expression wasobserved in 10.2% (No. 66; FIG. 9C), 27% (No. 1049, FIG. 9A) and 69.7%(No. 377; FIG. 9B) of the total population examined.

To investigate the capacity of self-renewal and clonogenic potential ofCD133⁺ cell, a single isolated CD133 positive cancer stem cell wasisolated by DAKOcytomation™ (DAKO, Carpinteria, Calif.) sorting andcultured in a defined serum-free NSC medium (Kabos et al., Exp. Neurol.,178:288-293, 2002) containing 20 ng/ml of basic fibroblast growth factor(bFGF, Peprotech, Rocky Hill, N.J.), 20 ng/ml of epidermal growth factor(EGF, Peprotech) and 20 ng/ml leukemia inhibitory factor (LIF, Chemicon,Temecula, Calif.). Single isolated CD133 positive cancer stem cells wereable to form neurospheres (FIG. 8A), and were demonstrated to have thecapacity for self-renewal and clonogenic potential (FIG. 8B), andsustained expression of CD133 in serum-free medium containing EGF/FGF(FIG. 8C).

Example 6 Human CD133 Positive Tumor Cells Express Markers Associatedwith Neural Precursors

To determine the expression of other genes in CD133 positive cells,CD133 positive cells and CD133 negative cells were obtained by FACSsorting as described in Example 5 and real-time PCR was used to analyzesome markers associated with neural precursors in these two populations.Total RNA was extracted from the isolated CD133 positive and CD133negative cells using an RNA4PCR™ kit (Ambion, Austin, Tex.) according tothe manufacturer's protocol. For cDNA synthesis, ˜1 μg total RNA wasreverse-transcribed into cDNA using Oligo dT primer and iScript™ cDNAsynthesis kit reverse transcriptase. cDNA was stored at −20° C. Geneexpression was quantified by real-time quantitative RT-PCR usingQuantiTect™ SYBR Green dye (Qiagen, Valencia, Calif.). DNA amplificationwas carried out using Icycler™ (BIO-RAD, Hercules, Calif.), and thedetection was performed by measuring the binding of the fluorescence dyeSYBR Green I to double-stranded DNA. All the primer sets were obtainedfrom Qiagen (see Table 1).

TABLE 2 Oligonucleotide primers sequences used for SYBRGreen real-time PCR Gene Forward Reverse Beta-5′-TTCTACAATGAGCTGCGTGTG-3′ 5′-GGGGTGTTGAAGGTCTCAAA-3′ actin(SEQ ID NO: 1) (SEQ ID NO: 2) CD133 5′-GCATTGGCATCTTCTATGGTT-3′5′-CGCCTTGTCCTTGGTAGTGT-3′ (SEQ ID NO: 3) (SEQ ID NO: 4) MSI15′-GAGACTGACGCGCCCCAGCC-3′ 5′-CGCCTGGTCCATGAAAGTGACG-3′ (SEQ ID NO: 5)(SEQ ID NO: 6) MELK 5′-CTTGGATCAGAGGCAGATGTTTGGAG-3′5′-GTTGTAATCTTGCATGATCCAGG-3′ (SEQ ID NO: 7) (SEQ ID NO: 8) PSP5′-GGCGGGGCAGTGCCTTTCAAA-3′ 5′-TGTTGGCTGCGTCTCATCAAAACC-3′(SEQ ID NO: 9) (SEQ ID NO: 10) CD90 5′-CGCTCTCCTGCTAACAGTCTT-3′5′-CAGGCTGAACTCGTACTGGA-3′ (SEQ ID NO: 11) (SEQ ID NO: 12) NESTIN5′-ATCGCTCAGGTCCTGGAA-3′ 5′-AAGCTGAGGGAAGTCTTGGA-3′ (SEQ ID NO: 13)(SEQ ID NO: 14) CD44 5′-AGAAGGTGTGGGCAGAAGAA-3′5′-AAATGCACCATTTCCTGAGA-3′ (SEQ ID NO: 15) (SEQ ID NO: 16) GLI15′-AGGGAGGAAAGCAGACTGAC-3′ 5′-CCAGTCATTTCCACACCACT-3′ (SEQ ID NO: 17)(SEQ ID NO: 18) CXCR4 5′-GATCAGCATCGACTCCTTCA-3′5′-GGCTCCAAGGAAAGCATAGA-3′ (SEQ ID NO: 19) (SEQ ID NO: 20) Bmi-15′-GGAGACCAGCAAGTATTGTCCTTTTG-3′ 5′-CATTGCTGCTGGGCATCGTAAG-3′(SEQ ID NO: 21) (SEQ ID NO: 22) PTCH1 5′-CGCCTATGCCTGTCTAACCATGC-3′5′-AAATGGCAAAACCTGAGTTG-3′ (SEQ ID NO: 23) (SEQ ID NO: 24) Snail5′-ACCACTATGCCGCGCTCTT-3′ 5′-GGTCGTAGGGCTGCTGGAA-3′ (SEQ ID NO: 25)(SEQ ID NO: 26) SIRT1 5′-ACTTGTACGACGAAGACGAC-3′5′-CAGAAGGTTATCTCGGTACC-3′ (SEQ ID NO: 27) (SEQ ID NO: 28) Survivin5′-TGCCTGGCAGCCCTTTC-3′ 5′-CCTCCAAGAAGGGCCAGTTC-3′ (SEQ ID NO: 29)(SEQ ID NO: 30) CIAP1 5′-CAGCCTGAGCAGCTTGCAA-3′5′-CAAGCCACCATCACAACAAAA-3′ (SEQ ID NO: 31) (SEQ ID NO: 32) CIAP25′-TCCGTCAAGTTCAAGCCAGTT-3′ 5′-TCTCCTGGGCTGTCTGATGTG-3′ (SEQ ID NO: 33)(SEQ ID NO: 34) NAIP 5′-GCTTCACAGCGCATCGAA-3′ 5′-GCTGGGCGGATGCTTTC-3′(SEQ ID NO: 35) (SEQ ID NO: 36) XIAP 5′-AGTGGTAGTCCTGTTTCAGCATCA-3′5′-CCGCACGGTATCTCCTTCA-3′ (SEQ ID NO: 37) (SEQ ID NO: 38) BCL-25′-CATGCTGGGGCCGTACAG-3′ 5′-GAACCGGCACCTGCACAC-3′ (SEQ ID NO: 39)(SEQ ID NO: 40) BCL-X_(L) 5′-TGCATTGTTCCCATAGAGTTCCA-3′5′-CCTGAATGACCACCTAGAGCCTT-3′ (SEQ ID NO: 41) (SEQ ID NO: 42) FLIP5′-CATCCACAGAATAGACCTGAAGACAA-3′ 5′-GCTTGGAGAACATTCCTGTAACTTG-3′(SEQ ID NO: 43) (SEQ ID NO: 44) BAX 5′-TGG AGCTGCAGAGGATGATTG-3′5′-GAAGTTGCCGTCAGAAAACATG-3′ (SEQ ID NO: 45) (SEQ ID NO: 46) BCRP-15′-TGGCTGTCATGGCTTCAGTA-3′ 5′-GCCACGTGATTCTTCCACAA-3′ (SEQ ID NO: 47)(SEQ ID NO: 48) MGMT 5′-CTGGCTGAATGCCTACTTCC-3′5′-CAACCTTCAGCAGCTTCCAT-3′ (SEQ ID NO: 49) (SEQ ID NO: 50) OCT45′-CCTGAAGCAGAAGAGGATCA-3′ 5′-CCGCAGCTTACACATGTTCT-3′ (SEQ ID NO: 51)(SEQ ID NO: 52)

Quantification of target gene mRNA as compared to an internal control(beta-actin) was performed by following a ΔC_(T) method. Anamplification plot that had been the plot of fluorescence signal vs.cycle number was drawn. The difference (ΔC_(T)) between the mean valuesin the duplicated samples of target gene and those of beta-actin werecalculated by Microsoft Excel and the relative quantified value (RQV)was expressed as 2^(−ΔCT). The relative expression of each gene wascompared to autologous CD133 negative cells. The results of the QT-PCTanalysis are presented in Table 3.

TABLE 3 Relative Expression of Genes in CD133+ Cancer Stem Cells No. 66No. 377 No. 1049 Gene name CD133− CD133+ CD133− CD133+ CD133− CD133+CD90 1 15.6 ± 0.66 1 12.8 ± 0.94  1 13.5 ± 0.75  CD44 1  5.7 ± 0.48 12.5 ± 0.22 1 2.8 ± 0.19 CXCR4 1 337.8 ± 29.2  1 251.5 ± 22.1  1 264.9 ±22.9  Nestin 1 21.4 ± 1.25 1 23.2 ± 1.65  1 22.1 ± 1.54  MSI 1  84 ± 7.61 75.4 ± 7.03  1 53.5 ± 6.2  MELK 1 1351 ± 95.8  1 467.7 ± 40.5  1 514.6± 45.6  GLI-1 1  46 ± 3.8 1 43 ± 4.5  1 49 ± 5.9  PTCH 1   16 ± 1.48 113.5 ± 0.85  1 14.3 ± 1.24  MGMT 1 32.4 ± 2.5  1 34.7 ± 2.9  1 56.3 ±4.2  BCRP1 1  6.5 ± 0.43 1 4.3 ± 0.25 1 4.8 ± 0.24 SIRT1 1  4.9 ± 0.34 14.2 ± 0.26 1 5.4 ± 0.29 FLIP 1  294 ± 25.5 1 157.6 ± 14.2  1 145.6 ±13.7  BCL-2 1 13.9 ± 0.95 1 4.9 ± 0.54 1 3.8 ± 0.54 BCL-XL 1  5.6 ± 0.391 3.2 ± 0.16 1 2.5 ± 0.14 cIAP1 1 39.0 ± 3.5  1 4.3 ± 0.53 1 5.6 ± 0.65cIAP2 1   3 ± 0.25 1 1.9 ± 0.12 1 1.7 ± 0.14 XIAP 1 21.9 ± 2.2  1 9.7 ±0.68 1 10.3 ± 0.91  NAIP 1 12.1 ± 0.75 1 6.4 ± 0.43 1 4.5 ± 0.62Survivin 1  1.6 ± 0.08 1 2.3 ± 0.18 1 2.4 ± 0.18 BAX 1 0.33 ± 0.03 10.49 ± 0.06  1 0.21 ± 0.05 

CD90, CD44, CXCR4, Nestin, Musashi-1 (Msi1), and maternal embryonicleucine zipper kinase (MELK) mRNA expression on CD133 positive cancerstem cells was upregulated by an average of 15.6, 5.7, 337.8, 2.14, 84,and 1351 fold, respectively, compared to the levels found on autologousCD133 negative tumor cells. mRNA levels for GLI1 and PTCH1 wereupregulated an average of 46 and 16 times, respectively, in CD133positive cells, as compared to CD133 negative cells. Furthermore, Bmi-1,phosphoserine phosphatase (PSP), SHH, OCT4 and Snail mRNA were expressedin CD133 positive cells derived from the three cell lines; none of thefive genes were detectable on CD133 negative cells. Additionally,anti-apoptotic genes were also upregulated (see Example 8).

Example 7 CD133 Positive Cancer Stem Cells are Resistant toChemotherapeutic Agents

To determine whether CD133 positive cancer stem cells were resistant tochemotherapeutic agents, the WST-1 Cell Proliferation Assay was used toexamine the drug sensitivity of CD133 positive cells and CD133 negativecells (both collected by FACS sorting from the three glioblastomapatients' primary cultured tumor cells as described above). CD133positive and negative cells were exposed to conventionalchemotherapeutic agents, temozolomide, carboplatin, VP-16 or taxol atvarious concentrations, for up to 48 hours in 10% FBS/F-12/DMEM culturemedium. Temozolomide was supplied by the Schering-Plough ResearchInstitute (Kenilworth, N.J.) and was dissolved in DMSO (Sigma ChemicalCo., St Louis, Mo.) at 100 mM stock solution. Carboplatin, etoposide(VP-16) and paclitaxel (Taxol) were obtained from Sigma-Aldrich (St.Louis, Mo.). CD133 positive cells isolated from No. 66 showed dramaticdrug resistance to the above four agents including temozolomide (FIG.10C), carboplatin (FIG. 10D), VP-16 (FIG. 10A), and Taxol (FIG. 10B) ascompared to autologous CD133 negative cells. CD133 positive cellsisolated from No. 377 showed significant resistance to carboplatin atall concentrations tested (FIG. 11A) and to VP-16 at 200 μM (FIG. 11B).CD133 positive cells isolated from No. 1049 showed significantresistance to carboplatin at 200 μM compared to autologous CD133negative cells (FIG. 11C). This example demonstrates increased drugresistance of CD133 positive cancer stem cells as compared to autologousCD133 negative cells.

Example 8 Anti-Apoptotic Genes are Upregulated in CD133 Positive CancerStem Cells

Real-time PCR of FACs-sorted CD133 positive and CD133 negative cells wasused to investigate the relative expression of multi-drug resistancegenes and genes related to inhibiting cell apoptosis between these twopopulations. BCRP1 has been demonstrated to play an important role inthe drug resistance of normal stem cells and tumor stem cells (Zhou etal., Nat. Med., 7:1028-34, 2001; Hirschmann-Jax et al., Proc. Natl.Acad. Sci. USA, 101:14228-33, 2004). Higher expression of BCRP1 (6.5fold) was found in CD133 positive cells as compared to that ofautologous CD133 negative cells. Furthermore, anti-apoptotic genes, suchas FLIP, BCL-2, and BCL-XL, were found at significantly higher levels(294, 13.9 and 5.6 times higher) in CD133 positive cells than in CD133negative cells. Also, inhibitor of apoptosis protein (IAPs) familymembers, XIAP, cIAP1, cIAP2, NAIP, and survivin were found at higherexpression levels on CD133 positive cells 21.9, 39.04, 3.03, 12.1, 6.73,and 1.6 times higher, respectively, than in CD133 negative cells. It hasbeen demonstrated that SIRT1 deacetylates the DNA repair factor Ku70,causing it to sequester the pro-apoptotic factor, Bax, away frommitochondria, thereby inhibiting stress-induced apoptotic cell death(Cohen et al., Science, 305:390-392, 2004). SIRT1 deacetylase mRNAexpression was increased 4.92 times in CD133 positive cells. Thepro-apoptotic gene BAX was decreased 3 times in CD133 positive cells ascompared to autologous CD133 negative cells. Thus, gene expressiondifferences were observed in CD133 positive cells as compared to CD133negative cells.

Example 9 Recurrent Glioblastomas Express Higher Levels of CD133

Malignant glioma is a highly recurrent tumor even after surgery,chemotherapy, radiation and immunotherapy. To address the potential roleof CD133 positive tumor cells in glioblastoma recurrence, the CD133expression upon first and second resection of tumor tissue from the samepatient were compared. We obtained primary tumor tissue from fivepathological confirmed grade IV astrocytoma (GBM) patients, and thenre-operated following radiation, chemotherapy, and/or immunotherapy toobtain recurrent tumor tissue. RNA extraction and RT-PCR was performedfor CD133 as described above. For all tested patients, CD133 expressionwas significantly higher in recurrent tumor tissue (relative expression˜2.5-19.1) than that in autologous primary tumor tissue (relativeexpression ˜1)(FIG. 12). This example implicates a resistant CD133positive tumor population in tumor recurrence.

Example 10 Immunization with Cancer Stem Cell Antigens IncreasedSurvival

A dendritic cell vaccine was generated using antigens from neurospheres,daughter cells, and monolayer cells. Immature dendritic cells weregenerated from the bone marrow of 6-12-week-old Fisher F344 rats aspreviously described (Talmor et al., Eur. J. Immunol., 28:811-817,1998). Briefly, bone marrow was harvested from the femoral and tibialmarrow cavities and cultured in RPMI 1640 media supplemented with 10%fetal bovine serum (Gemini Biotechnologies, Calabasas, Calif.), 1%Penicillin/Streptomycin (Invitrogen, Carlsbad, Calif.), 50 ng/mlrecombinant rat GM-CSF and 100 ng/ml recombinant rat IL-4 (R & DSystems, Minneapolis, Minn.). Cultures were fed every 2 days by removing75% of the media and replacing it with fresh media containing cytokines(this washed away most of the lymphocytes and granulocytes). Todetermine the percentage of immature dendritic cells generated andcultured from the bone marrow of Fisher rats after 1 week of exposure toGM-CSF and IL-4, FACS analysis was run on cells immunostained withantibodies for CD86 (costimulatory marker B7-1; DC marker), CD80(costimulatory marker B7-2; DC marker), CD3 (T cell marker), or MHC II(DC marker). The dendritic cell populations obtained were positive forCD86, CD80, and MHC II, while negative for CD3.

Soluble peptides were generated for dendritic cell pulsing by celllysis. 9L neurospheres, daughter cells, and monolayer (adherent) cellswere processed in the laboratory to produce a single cell suspension.The cells were then lysed by 4 to 5 freeze cycles (on liquid nitrogen)and thaw cycles (room temperature). Lysis was monitored by lightmicroscopy, and larger particles were removed by centrifugation (10minutes at 600×g). The supernatants were passed through a 0.2 μm filter,and protein concentration was determined by BioRad protein assay andaliquots frozen at −80° C. until use.

To establish intracranial tumors, adult Fisher F344 rats werestereotactically inoculated in the right corpus striatum (from bregma inmm: anterior-posterior +1 mm; medial-lateral −3 mm; dorsal-ventral −5mm) with 25,000 9L-luciferase tumor cells as described above. Forvaccination of the rats, freshly cultured immature dendritic cells werecocultured overnight for 24 hours with 80-100 μg of cell lysate one dayprior to the vaccination at days 7, 14, and 21 post-operatively.Vaccinations were given subcutaneously in the flanks on days 7, 14, and21 with 50,000 DCs pulsed with antigens from either 9L neurospheres(NS), daughter cells (DtC), monolayer (adherent) cells (AC), or salinecontrol. The animals were followed for survival and euthanized whenterminal neurological signs developed, for example, inability to accessfood, water, seizure activity, weakness, and paralysis.

Tumor bearing rats injected with three successive vaccines (once perweek) of dendritic cells pulsed with control, AC, DtC, or NS antigens,had median survival dates of 26.5, 32, 29 and 50 days, respectively(FIG. 13). Kaplan-Meier analysis showed that rats treated with 9Lneurosphere lysate-pulsed DC had significantly longer survival time thaneach of the other groups (p=0.0015).

Example 11 DCs Pulsed with Cancer Stem Cell Antigens Induced a StrongCytotoxic T Cell Response Against 9L Tumor Cells

To determine whether the relative protective effect of NS-DC vaccinationon survival is due to tumor-specific immunity, a cytotoxic T lymphocyte(CTL) assay was performed. Spleens were removed on day 28post-intracranial 9L-luciferase tumor cells implantation from groups ofrats treated with either control or 9L peptide-pulsed dendritic cells.Splenocytes were isolated and re-stimulated in vitro, as described(Wunderlich et al., “Assays for T-cell function,” In: Coligan et al.,eds., Current Protocols in Immunology, New York, N.Y., John Wiley &Sons, Inc; 1997:3.11.1-3.11.20; Ehtesham et al., J. Immunother.,26:107-116, 2003) with either irradiated (10,000 rads) 9L adherent cells(AC), daughter cells (DtC) or neurospheres (NS). The effector: target(E:T) ratio was 6:1. Re-stimulated cells were cultured in RPMI-1640 with10% fetal bovine serum, 1% penicillin-streptomycin, and 1% HEPES in6-well flat-bottom plates in a humidified 37° C., 5% CO₂ incubator.Cells were incubated for 11 days with addition of IL-2 (300 units/mL)every 3 days. After 11 days of re-stimulation, the cells were re-exposedto their initial target (i.e., irradiated 9L AC, DtC, or NS). 24 hourslater, culture media was harvested from the remaining cells and used forIFNγ protein quantification by ELISA (Ehtesham et al., J. Immunother.,26:107-116, 2003). Re-stimulated splenocytes from rats treated with NSpulsed DCs showed significantly higher IFNγ release in response toexposure to tumor cell targets than re-stimulated splenocytes from ratstreated with AC or DtC (FIG. 14), indicating that a higher cytotoxic Tcell response was obtained in the NS-DC vaccinated rats. These data werealso confirmed by PCR for IFNγ levels (data not shown). The higher IFNγresponse in the NS-DC vaccinated group corresponds to the highersurvival rate observed in the same group. These data show that a cancerstem cell antigen vaccine targets tumor cells more potently thanvaccines based on tumor antigens from daughter cells, or cellpopulations that are not enriched for cancer stem cells.

Example 12 Intracranial T-Cell Infiltration is Associated with ProlongedSurvival

A representative number of brains from DC-DtC and DC-NS vaccinated ratswere carefully removed and post-fixed in 4% paraformaldehyde. Coronalsections of 20 μm were cut on a cryostat and blocked with normal horseserum for 1 hour. Slides were then incubated with anti-CD4 (clone OX-38monoclonal antibody diluted in 1:200 in PBS) for 2 hours at roomtemperature, followed by a 20 minute incubation at room temperature withthe linking antibody (BioGenex biotinylated anti-mouse immunoglobulin).After washing in PBS, the labeled moiety (BioGenex Horse RadishPeroxidase-conjugated streptavidin) was added for 20 minutes at roomtemperature. DAB (3,3′-diaminobenzidine) was used as the chromogen. Foranalysis of CD8 expression, slides were incubated overnight withAnti-CD8 alpha Chain, clone OX-8 antibody (Chemicon).

Immunohistochemical assessment of brain sections from rats vaccinatedwith dendritic cells pulsed with NS vaccinated showed that there was arobust infiltration of CD4⁺ (FIG. 15B) and CD8⁺ lymphocytes that was notobserved in the brain sections obtained from rats vaccinated withdendritic cells pulsed with daughter cell antigens (FIG. 15A). Thisinfiltration of T cells correlates with increased survival of ratsvaccinated with NS pulsed DCs.

Example 13 Cancer Stem Cells Express MHC, Unlike Normal Stem Cells

Five glioblastoma multiforme (GBM) cancer stem cells (CSC) weregenerated by the methods described in Yuan et al. (Oncogene,23:9392-9400, 2004). Briefly, tumor specimens were obtained within halfan hour of surgical resection from five adult GBM patients, as approvedby the Institutional Review Boards at the Cedars-Sinai Medical Center.Tumor tissue was washed, minced, and enzymatically dissociated (Reynoldset al., Science, 255:1707-10, 1992). Tumor cells were resuspended inDMEM/F12 medium containing 10% fetal bovine serum (FBS) as growth mediumand plated at a density of 2×10⁶ live cells per 75 cm² flask. The cellsattached and grew as a monolayer in flasks. All the five monolayergrowing adult GBM cells were switched into a defined serum-free NSCmedium (Reynolds et al., Science, 255:1707-10, 1992) containing 20 ng/mlof basic fibroblast growth factor (bFGF, Peprotech, Rocky Hill, N.J.)and 20 ng/ml of epidermal growth factor (EGF, Peprotech). Normal humanfetal neural stem cells (NSC) were cultured in the same definedserum-free medium as for cancer stem cells. CSC and NSC cells werestained by FITC-HLA-A,B,C antibody and isotype control antibody (BDBioscience, San Diego, Calif.) and analyzed by flow cytometry.Representative CSC and NSC results were shown in the FIGS. 17A-17B and18A-18B, respectively.

HLA expression was seen in 5 of 5 cancer stem cells from differentpatients. CSCs expressed high levels of HLA-A,B,C, however, NSC did notexpression MHC class I (HLA-A,B,C) antigens on the surface. Thisunexpected result indicates that specific cancer stem cell antigens oncancer stem cells can be targeted using vaccines that generate T cellsthat recognize and kill cancer stem cell antigens in the context of MHC.Therefore cancer stem cells will be targeted, whereas normal stem cellswill not.

Example 14 Isolation of CD133 T Cell Epitopes

A nucleic acid encoding a portion of CD133 extracellular domain 1 (aminoacid residues 116-270 of SEQ ID NO:53) is used in cloning and expressionon the surface of DC. DC are transfected with CD133-1 cDNA construct orempty vector (mock) for 48 hours. The successful transfection ofDC-CD133 cells is identified either by anti-CD133 monoclonal antibodyusing flow cytometry or by EGFP cloned vector under fluorescentmicroscopy.

Antigen presenting cells with CD133 receptor are stimulated by CD133 Tcell epitopes. Overlapping peptides of 8-10 amino acids of residues325-350 of SEQ ID NO:53 are produced as MHC class I epitopes. Similarly,overlapping peptides of 13-20 amino acid of residues 325-350 areproduced as MHC class II epitopes. Stimulated APC using the peptideepitopes lead to enhanced production of CD8 T cells targeted to CD133molecular on stem/progenitor cells in brain tumors.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating glioblastoma in a patient,the method comprising: obtaining a population of dendritic cells;contacting the dendritic cells with a glioblastoma cancer stem cellantigen composition consisting essentially of antigens derived fromisolated glioblastoma cancer stem cells under conditions such that thedendritic cells present glioblastoma cancer stem cell antigens; andadministering to a patient a composition comprising the dendritic cells.2. The method of claim 1, wherein the dendritic cells are autologous. 3.The method of claim 1, wherein the dendritic cells are allogeneic. 4.The method of claim 1, wherein the glioblastoma cancer stem cell antigencomposition is obtained from glioblastoma cancer stem cells isolatedfrom a glioblastoma tumor.
 5. The method of claim 1, wherein theglioblastoma cancer stem cell antigen composition comprises a lysate ofglioblastoma cancer stem cells.
 6. The method of claim 1, wherein theglioblastoma cancer stem cell antigen composition comprises an acideluate of glioblastoma cancer stem cells.
 7. The method of claim 5,wherein the glioblastoma cancer stem cells express CD133.
 8. The methodof claim 1, wherein the glioblastoma cancer stem cell antigencomposition comprises one or more isolated peptides of CD133, CD90,CD44, CXCR4, Nestin, Musashi-1 (Msi1), maternal embryonic leucine zipperkinase (MELK), GLI 1, PTCH1, Bmi-1, phosphoserine phosphatase (PSP),Snail, OCT4, BCRP1, MGMT, Bcl-2, FLIP, BCL-XL, XIAP, cIAP1, cIAP2, NAIP,or survivin.
 9. The method of claim 8, wherein the one or more isolatedpeptides are synthetic.
 10. A method of treating glioblastoma in apatient, the method comprising: administering to a patient a compositionconsisting essentially of dendritic cells that present glioblastomacancer stem cell antigens.
 11. The method of claim 10, wherein theglioblastoma cancer stem cell antigens are obtained from glioblastomacancer stem cells isolated from a glioblastoma tumor.
 12. The method ofclaim 10, wherein the glioblastoma is a recurrent glioblastoma.
 13. Themethod of claim 1, wherein the glioblastoma is a recurrent glioblastomacancer.
 14. The method of claim 1, wherein the patient is human.
 15. Themethod of claim 10, wherein the patient is human.